Investigation of the Immunomodulatory effect of Berberis vulgaris on core-pulsed dendritic cell vaccine

Abstract

Background: Virus-induced dendritic cells (DCs) functional deficiency leads to sub-optimal initiation of adaptive immune responses and consequently chronic infection establishment. The present study reports an advanced hepatitis C virus (HCV) therapeutic vaccine model based on In vivo enrichment of DCs with barberry ethanolic crude extract (BCE) then pulsing them with HCV core protein.

Methods: DCs were enriched by BCE intravenous injection in BALB/c mice. Vaccine efficiency was assessed by flow cytometric analysis of splenocytes of immunized mice, cytokine profiling, cytotoxic T lymphocyte assay, and humoral immune response assessment.

Results: There was no significant difference in surface phenotypic characterization of splenocytes from mice immunized with non-BCE-enriched-core-pulsed DCs (iDcs-core) compared to those from mice injected with RPMI-1640 medium. However, splenocytes from mice immunized with BCE-enriched-core-pulsed DCs showed 197 % increase in CD16+ population, 33 % increase in MHCII(+) population, and 43 % decrease in CD3(+) population. In iDCs-core group, 57.9 % greater anti-core cytotoxic T lymphocyte activity, up-regulation in interferon gamma and interleukin (IL) -12 expression, and down-regulation in IL-4 and IL-10 were recorded. Moreover, sustained specific anti-core antibodies were detected only in sera of the same group.

Conclusions: results indicate that BCE-enriched-core-transduced DCs may serve as a new model for immunotherapy of HCV chronic infection.

Keywords: Berberis vulgaris; IL 12; IL 4; INF-γ; Th-1.

Fig. 1

Effect of immunization with different subsets of DCs on surface marker expression in splenocytes of immunized mice. Splenocytes of immunized mice with BCE-induced-core pulsed dendritic cells, in a 3-dose immunization schedule at 4 weeks intervals, shows an elevated CD16⁺and MHCII⁺ populations when compared to RPMI - 1640 group. Spleens of immunized mice were dissociated by gentleMACS dissociator, as described in Materials and Methods, into a single-cell suspension. Freshly isolated splenocytes were stained with MHCII-FITC, Fcγ RIII/CD16-APC, and CD3-PE monoclonal antibodies and analyzed by flow cytometry. Data are shown as the percentage of splenocytes bearing surface antigen over total splenocytes population. * indicates a significant difference with RPMI group. Results are means ± SD of splenocytes of six mice. (PRPMI - 1640 group that received complete medium, nDCs group that received non-BCE-induced DC, nDCs-core group that received non-BCE-induced DCs transduced with core protein, iDCs group that received BCE-induced DCs, iDCs-core group that received BCE-induced DCs transduced with core protein)

Fig. 2

Effect of mice immunization on the expression of splenic INF-γ, IL-12p40, IL-4 and IL-10 respectively. Cytokine profile of splenocytes of mice immunized with BCE-induced-core-pulsed DCs exhibits a great shifting towards up-regulating Th1 cytokine profile and down-regulating Th2 cytokine profile. Specific PCR amplification of reverse-transcribed total RNA (1 μg) from 1 × 10⁶ of splenocytes of mice immunized with different subsets of DCs for 4 weeks

Fig. 3

Cytolytic protein expression of INF-γ after mice immunization. Mice immunized with iDCs or iDCs-core cells increases intrasplenic IFN-γ production in their splenocytes when compared to RPMI group and/or nDCs and nDCs-core groups. Cytoplasmic contents of cytokines protein expression of INF-γ was assessed from 1 × 10⁶ of splenocytes using ELISA as previously mentioned at material and methods. Data are shown as the percentage of splenocytes bearing surface antigen over total splenocytes population. * indicates a significant difference with RPMI- 1640 group. Results are means ± SD of splenocytes of six mice. # indicates a significant difference within nDCs, and nDCs-core groups

Fig. 4

Splenocytes of iDCs-core group have a good T lymphocyte suppression capacity only on EL4-core cells. Splenocytes of immunized mice (E) were co-cultured with EL-4 or EL-4-core cells (T) at a ratio of 1:1.6 E:T. EL-4 cells were transduced with core recombinant peptides using BioPORTER quickEase protein delivery Kit as previously described. Co-cultured cells were incubated at 37 °C for 4 h and their cytotoxicity was assessed using MTT viability test and LDH release. Data are shown as the percentage of splenocytes bearing surface antigen over total splenocytes population. * indicates a significant difference with RPMI group. Results are means ± SD of splenocytes of six mice. # indicates a significant difference within nDCs, and nDCs-core groups

Fig. 5

Antigen-specific IgG antibodies generated in mice by immunization with DCs. Humoral response assessment revealed potent and specific anti-core IgG antibodies (total IgG) only in iDCs-core group. Detection of igG antibody responses to the HCV core protein in immunized mice by ELISA. Pooled sera (1:50 dilution) from each group were incubated in wells coated with recombinant HCV core protein (NS3 protein was used as a negative control) and detected by a goat anti-mouse secondary antibody (immuno- globulin) conjugated to alkaline phosphatase. The results are expressed as the optical densities (O.D.) at 405 nm. * indicates a significant difference among groups. Results are means ± SD of sera of six mice