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Differential activity of human leukocyte extract on systemic immune response and cyst growth in mice with Echinococcus multilocularis infection after oral, subcutaneous and intraperitoneal routes of administration

D. Ciglanová
D. Ciglanová
, 
Z. Jurčacková
Z. Jurčacková
, 
D. Mudroňová
D. Mudroňová
, 
E. Dvorožňáková
E. Dvorožňáková
 and 
G. Hrčková
G. Hrčková
   | Dec 30, 2022
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Article Category: Research Article
Published Online: Dec 30, 2022
Page range: 341 - 356
Received: Oct 11, 2022
Accepted: Dec 04, 2022
DOI: https://doi.org/10.2478/helm-2022-0038
Keywords
© 2022 D. Ciglanová, Z. Jurčacková, D. Mudroňová, E. Dvorožňáková, G. Hrčková, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

The effects of DLE administration by PO, SC and IP routes on the parasite cysts weights in the peritoneal cavities of infected mice. The numbers represent means ± SD of (n = 5 mice/ group). Significantly different values between infected and treated mice are indicated by connecting line, **p < 0.01.
The effects of DLE administration by PO, SC and IP routes on the parasite cysts weights in the peritoneal cavities of infected mice. The numbers represent means ± SD of (n = 5 mice/ group). Significantly different values between infected and treated mice are indicated by connecting line, **p < 0.01.

Fig. 2 A-B

The effects of DLE administration by PO, SC and IP routes on: A – total numbers of leukocytes in the peripheral blood; B – proportions of the lymphoid populations within WBC. C – dot plot showing forward and side analysis. Data are expressed as the means ± SD. Significantly different values were calculated between a – control and infected and infected treated mice; b – infected and treated mice, and are indicated by connecting lines,* p < 0.05, **p < 0.01, *** p < 0.001, ns – nonsignificant.
The effects of DLE administration by PO, SC and IP routes on: A – total numbers of leukocytes in the peripheral blood; B – proportions of the lymphoid populations within WBC. C – dot plot showing forward and side analysis. Data are expressed as the means ± SD. Significantly different values were calculated between a – control and infected and infected treated mice; b – infected and treated mice, and are indicated by connecting lines,* p < 0.05, **p < 0.01, *** p < 0.001, ns – nonsignificant.

Fig. 3 A-D

The proportions of lymphoid cells subpopulations in the peripheral blood of control, infected and infected and treated mice were analyzed by flow cytometry. A – lymphoid populations were gated on CD3+T lymphocytes and B – B220+ B lymphocytes; C – CD4+T helper and D – CD8+ cytotoxic lymphocytes were gated within CD3+ cells. Data are expressed as the means ± SD. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines,* p < 0.05, **p < 0.01, *** p < 0.001, ns-nonsignificant.
The proportions of lymphoid cells subpopulations in the peripheral blood of control, infected and infected and treated mice were analyzed by flow cytometry. A – lymphoid populations were gated on CD3+T lymphocytes and B – B220+ B lymphocytes; C – CD4+T helper and D – CD8+ cytotoxic lymphocytes were gated within CD3+ cells. Data are expressed as the means ± SD. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines,* p < 0.05, **p < 0.01, *** p < 0.001, ns-nonsignificant.

Fig. 4 A-B

The effects of DLE administration on the A – phagocytic activity and B – MFI of monocytes and neutrophils in the peripheral blood of mice. Significantly different values were calculated between a – control and infected and infected+ treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, ns – nonsignificant.
The effects of DLE administration on the A – phagocytic activity and B – MFI of monocytes and neutrophils in the peripheral blood of mice. Significantly different values were calculated between a – control and infected and infected+ treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, ns – nonsignificant.

Fig. 5 A-F

Phenotypic analysis of myeloid-derived populations in peripheral blood of mice was performed by flow cytometry. Cells in control, infected and infected and treated mice were gated on A – CD11b+ cells and B – CD11b+MHCIIhigh monocytes, C-D – the representative histograms showing expression of MHCII in infected and PO-treated group. E – the proportions of CD11b-SiglecF+ eosinophil-like cells, F – the proportions of CD11b+SiglecF+ eosinophils. Significantly different values were calculated between a/control and infected and infected+treated mice; b/ infected and treated mice, and are indicated by connecting lines. * p < 0.05, **p < 0.01, *** p < 0.001; ns-nonsignificant.
Phenotypic analysis of myeloid-derived populations in peripheral blood of mice was performed by flow cytometry. Cells in control, infected and infected and treated mice were gated on A – CD11b+ cells and B – CD11b+MHCIIhigh monocytes, C-D – the representative histograms showing expression of MHCII in infected and PO-treated group. E – the proportions of CD11b-SiglecF+ eosinophil-like cells, F – the proportions of CD11b+SiglecF+ eosinophils. Significantly different values were calculated between a/control and infected and infected+treated mice; b/ infected and treated mice, and are indicated by connecting lines. * p < 0.05, **p < 0.01, *** p < 0.001; ns-nonsignificant.

Fig. 6

The proportions of: A – lymphoid and B – myeloid cell populations in the spleens of control, infected and infected and treated mice were analysed by flow cytometry. Data are expressed as means ± SD. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, **p < 0.01, *** p < 0.001.
The proportions of: A – lymphoid and B – myeloid cell populations in the spleens of control, infected and infected and treated mice were analysed by flow cytometry. Data are expressed as means ± SD. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, **p < 0.01, *** p < 0.001.

Fig. 7 A-B

The proliferation index of: A – Con A stimulated T lymphocytes and B – LPS stimulated B lymphocytes, isolated from the spleens of control, infected and infected and treated mice. Data are expressed means ± SD of triplicates/mouse/group after 70 hrs of incubation ex vivo. Cell proliferation was evaluated using BrdU Cell Proliferation ELISA Kit. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, **p < 0.01, *** p < 0.001.
The proliferation index of: A – Con A stimulated T lymphocytes and B – LPS stimulated B lymphocytes, isolated from the spleens of control, infected and infected and treated mice. Data are expressed means ± SD of triplicates/mouse/group after 70 hrs of incubation ex vivo. Cell proliferation was evaluated using BrdU Cell Proliferation ELISA Kit. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, **p < 0.01, *** p < 0.001.

Fig. 8 A-C

The production of cytokines by Con A stimulated T lymphocytes isolated from the spleens of control, infected and infected and treated mice ex vivo. Lymphocytes were cultured for 70 hrs and cytokines were determined in the supernatants by ELISA tests. Data are expressed means ± SD of duplicates/mouse/group in pg/ml. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, **p < 0.01, *** p < 0.001.
The production of cytokines by Con A stimulated T lymphocytes isolated from the spleens of control, infected and infected and treated mice ex vivo. Lymphocytes were cultured for 70 hrs and cytokines were determined in the supernatants by ELISA tests. Data are expressed means ± SD of duplicates/mouse/group in pg/ml. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.* p < 0.05, **p < 0.01, *** p < 0.001.

Fig. 9 A-F

Relative m-RNA expression of cytokines IFN-γ, IL-4 and TGF-β and corresponding transcriptions factors T bet, GATA3 and FoxP3 (shown as the fold change) was examined by qRT-PCR in the samples of unstimulated lymphocytes isolated from the spleens of control, infected and infected and treated mice. Relative gene expression was calculated by 2-ΔΔCt method using data for control healthy mice as calibrator following normalisation to GAPDH. Data are expressed means ± SD of duplicates/mouse/group. Significantly different values between infected and treated mice are indicated by connecting line, *p < 0.05, **p < 0.01, ***p < 0.001.
Relative m-RNA expression of cytokines IFN-γ, IL-4 and TGF-β and corresponding transcriptions factors T bet, GATA3 and FoxP3 (shown as the fold change) was examined by qRT-PCR in the samples of unstimulated lymphocytes isolated from the spleens of control, infected and infected and treated mice. Relative gene expression was calculated by 2-ΔΔCt method using data for control healthy mice as calibrator following normalisation to GAPDH. Data are expressed means ± SD of duplicates/mouse/group. Significantly different values between infected and treated mice are indicated by connecting line, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 10

The concentration of NO determined in the culture supernatants of LPS-stimulated adherent myeloid cells (1x106) isolated from the spleens of control, infected and infected and treated mice ex vivo. NO was determined as nitrite (NO2) using Griess reagent. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.**p < 0.01, *** p < 0.001.
The concentration of NO determined in the culture supernatants of LPS-stimulated adherent myeloid cells (1x106) isolated from the spleens of control, infected and infected and treated mice ex vivo. NO was determined as nitrite (NO2) using Griess reagent. Significantly different values were calculated between a – control and infected and infected+treated mice; b – infected and treated mice, and are indicated by connecting lines.**p < 0.01, *** p < 0.001.

List of oligonucleotides and their sequences.

Gene Orientation Sequence
GAPDH forwardreverse 5′-AGGTCGGTGTGAACGGATTTG-3‘5′-TGTAGACCATGTAGTTGAGGTCA-3‘
IFN-γ forwardreverse 5′-TCAAGTGGCATAGATGTGGAAGAA-3‘5‘- TGGCTCTGCAGGATTTTCATG-3‘
TGF-β forwardreverse 5′-TGACGTCACTGGAGTTGTACGG-3′5′-GGTTCATGTCATGGATGGTGC-3′
IL-4 forwardreverse 5′-TCAACCCCCAGCTAGTTGTC-3′5′-TTCAAGCATGGAGTTTTCCC-3′
FoxP3 forwardreverse 5′-AATAGTTCCTTCCCAGAG-3′5′-GATTTCATTGAGTGTCCT-3′
T-bet forwardreverse 5′-GCCAGGGAACCGCTTATATG-3′5′-TGGAGAGACTGCAGGACGAT-3′
GATA3 forwardreverse 5′-GAAGGCATCCAGACCCGAAAC-3′5′-ACCCATGGCGGTGACCATGC-3′

The total numbers of white blood cell subpopulations in 1 ml of peripheral blood.

Routes of DLE administration
Groups of mice/ cell subpopulations CTRL INF PO SC IP
White blood cells 8.72 ± 1.17 6.10 ± 0.46 7.02 ± 0.82 5.65 ± 0.67 7.06 ± 1.05
Lymphocytes/WBC 5.86 ± 0.94 1.79± 0.55 3.54±0.67 2.61±0.46 2.66 ± 0.49
CD3+/lymphocytes 3.41 ± 0.62 1.41 ± 0.43 2.42 ± 0.51 2.02 ± 0.38 2.04 ± 0.23
B220+/lymphocytes 2.33 ± 0.29 0.34 ± 0.13 1.03 ± 0.24 0.56 ± 0.09 0.52 ± 0.11
CD11b+/WBC 3.71 ± 0.71 4.21 ± 0.55 4.55 ± 0.85 3.95 ± 0.43 5.23 ± 0.99
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