Impaired cellular functions of immune system upon HCV
Impairment of the maturation process in dendritic cells (DCs) is one of the mechanisms responsible for immune evasion of hepatitis C virus (HCV) [
6]. A study reported that myeloid DCs have an up-regulated cytotoxic activity to kill T cells during HCV chronic infection, which represents a novel mechanism of HCV evasion [
22]. HCV core protein can interact with multiple cellular factors, and regulate expression of cellular genes and control signaling pathways of different cell types, including DCs [
25,
26].
Several studies have shown that core protein may have a pro-apoptotic activity by affecting the tumor necrosis factor receptor (TNFR) signaling pathway. Since lymphotoxin β receptor (LTβR) is involved in apoptotic signaling, this suggests that core protein may have an immunomodulatory function and may also play a critical role in the establishment of HCV persistence and in disease pathogenesis [
26,
27]. Therefore, the cytoplasmic domain of LTβR and TNFR1 signaling is important for the microenvironment that allows interactions of lymphocytes with antigen presenting cells and for B cell migration and differentiation into antibody-producing cells. Impairment of these functions may interfere with the elimination of infected cells and neutralization of virus. [
28] Core protein reduces sensitivity to tumor necrosis factor (TNF) and activates nuclear factor kappa B (NFkB), thus inhibiting apoptosis either constitutively or in response to cytokines [
29]. HCV can infect T cells, so any decrease in the apoptosis thresholds by core may impair their activation and cytotoxic functions. Since HCV can infect lymphocytes, increasing their sensitivity to apoptotic stimuli affects their activation and immune functions. In chronic infection, few HCV specific cytotoxic T lymphocytes (CTL) are found in the peripheral blood, which could be due to an abnormal death of activated CTL [
30,
31].
Interaction of virus particle with cell surface molecules, such as CD81, may modulate cell signaling. CD81 is widely expressed and found on natural killer (NK) cells. Inhibition of interferon-gamma (INF-γ) production by NK cells could alter the development of a T helper 1 (Th1) response and favor a T helper 2 (Th2). Inhibition of the innate immune response early after infection could confer a growth advantage to HCV that could not be controlled by the adaptive immune response. The inefficient NK cell response could allow the selection of escape variants [
32]. Compared to monocyte-derived DCs from healthy donors, DCs from patients with chronic HCV infection showed an impaired ability to stimulate allogeneic T cells and to produce interferon (INF) [
33]. This impaired maturation of DCs has been correlated with persistent HCV infection, [
34,
35] and that’s the main scope of the current study.
Role of BCE on DCs enrichment
A number of studies described a significant down-regulation of DCs function in HCV infected patients, shifting the Th1/Th2 balance towards Th2 up-regulation [
7], so a therapeutic DCs vaccine may hold its promises by ex vivo maturation and stimulation of DCs, because DCs of chronically infected patients are under a negative regulation of the virus itself. It was demonstrated that treatment of macrophages and DCs with berberine; a benzodioxoloquinolizine alkaloid present in
Berberis vulgaris plant, significantly induced interleukin (IL) -12 production in a dose-dependent manner, leading to the inhibition of Th2 cytokine profile in CD4
+ T cells which was also observed in our results (Table
1) with a decrease in Th1 cytokines (IL 10, and IL4) in both protein and mRNA level [
13‐
15,
17‐
36] (
https://dragonherbarium.com/products/barberry-root-bark-c-s-wc-berberis-vulgaris). In the present study, results of phenotypic characterization of immunized mice’s splenocytes proved that barberry crude extract (BCE) was the most powerful inducer for splenic cells to express DCs surface markers with acquiring the functional characteristics associated with DCs. The maximal increase CD11c
+ cells of animal model (A) after BCE intravenous injection by 10 folds with an increase in MHC II surface marker which indicate an increase in antigen presenting capacity (Table
1).
Table 1
Phenotyping analysis of DCs
CD11c (%) | 16 ± 0.9 | 1.5 ± 0.01 |
MHCII (%) | 13 ± 0.4 | 8 ± 0.3 |
IL4 (fold) | 0.31 ± 0.01 | 0.61 ± 0.002 |
IL 10 (fold) | 0.27 ± 0.03 | 0.73 ± 0.002 |
IL 12 (fold) | 0.74 ± 0.005 | 0.2 ± 0.003 |
INF (fold) | 1.3 ± 0.05 | 0.56 ± 0.002 |
IL 12 (Pg/ml) | 4.5 ± 0.3 | 1.2 ± 0.1 |
INF-gamma (Pg/ml) | 35 ± 3.2 | 22.5 ± 1.2 |
CD11c increase in DCs treated with BCE had exceeded the conventional DCs stimulators such as recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant fms-like tyrosine kinase receptor-3 (Flt3) ligand according to a study by Berhanu el al. [
37] These workers found that BALB/c mice subcutaneous injection with GM-CSF had expanded splenic CD11c
+ cells by about one fold, and Flt3 ligand injection expanded them by about five folds, whereas the combinatory GM-CSF and Flt3 ligand injection had expanded CD11c
+ cells by 9 folds.
CD16-mediated signal transduction promotes induction of maturation of immature DCs and CD16-mediated antigen uptake potently enhances antigen presentation [
20,
21]. CD16
+ cells in iDCs-core splenocytes exceeding those in RPMI - 1640 group by approximately 3 folds, reflected an up-regulation in pro-inflammatory cytokines that might represent DCs precursors. Moreover, it might indicate an elevated phagocytic activity and antigen presenting capacity of the same group [
38]. Elevated expression of major histocompatibility (MHC) II surface antigen on splenocytes of iDCs-core group identified a high regulation of antigen presentation, DCs morphology, and expression of co-stimulatory molecules which was supported by Banchereau, et al. [
22] Although CD16 is not DCs specific, it was found on a limited number of other cell types like NK cells and macrophages [
39]. However, co-expression of CD16 with MHC-II has been previously described by Haverson [
40] as being characteristic for DCs in the porcine intestinal lamina propria. While another important marker for splenic DCs is CD11c, since the injected cells used for immunization were solely CD11c
+. Low CD3
+ cells from immunized mice may be attributed to the architectural changes in the T cell receptor (TCR):CD3 complexes induced by MHCII:core protein ligation after immunization with DCs induced by BCE and pulsed with core protein (as MHCII T cell interaction complex causes internalization and degradation of CD3 antigen). The rate of TCR internalization did not increase significantly, while the binding of a peptide: MHCII ligand to the TCR promoted significant down-regulation of TCR complexes. This trend suggested that the binding of the ligand blocks TCR complexes from returning to the cell surface rather than increasing the rate of internalization of TCR complexes expressed on the cell surface [
41].
The present phenotypic reports clearly emphasized the important role BCE on DCs enrichment and maturation. It also emphasized that immunization with BCE-induced DCs might counteract DCs defects that took place upon HCV infection.
HCV core protein triggered monocyte-derived IL-10 productions. This cytokine, in turn, has led to DCs apoptosis and impaired production of interferon-alpha (INF-α), thus closely resembling the DCs defects seen in chronically HCV-infected patient. Multiple immune defects have been described in patients with chronic HCV infection that might be linked to the reduced INF-α production, including insufficient response of CTL, low activity of NK cells, and production of antibody with low neutralizing capacity [
42,
43]. Most antigens and vaccines trigger both B and T cell responses, such that there is no rationale in opposing antibody production (humoral immunity) and T cell responses (cellular immunity). In addition, Th cells are required for most antibody responses, while antibodies exert significant influence on T cell responses to intracellular pathogen [
44,
45]. Vaccine–induced immune effectors are essentially antibodies produced by B lymphocytes and capable of binding specifically to a toxin or a pathogen peptide. Other potential effectors are CTL that may limit the spread of infectious agents by recognizing and killing infected cells or secreting specific antiviral cytokines. The generation and maintenance of both B and CTL responses is supported by growth factors and signals provided by Th cells. These effectors are controlled by regulatory T cells that are involved in maintaining immune tolerance.
Anti-core cellular response
The communication between DCs and T cells in this area of host–pathogen interaction seems to be a dialogue rather than a monologue in which mature DCs respond to T cells as well. In the process of studying DCs vaccine, it is worth mentioning that INF-γ, IL-12, IL-4, IL-10, and other cytokines had a crucial role in manipulating a potent anti-HCV cellular immune response after vaccination. Up-regulation of Th1 cytokines (e.g. IL-12, INF-γ) and down-regulation of Th2 cytokines (e.g. IL-4, IL-10) and nitric oxide (NO) were the major targets to retain the th1/th2 balance corrupted by HCV viral infection and have an adequate anti-HCV cellular immune response. Interferons are key players of the innate immune response to virus infection. The production of INF-γ (type II INF) is restricted to cells of the immune system, such as NK cells, macrophages, and T cells [
46]. IL-12, which is known to be the primary stimulator of NK cells and INF-γ, induce Th1 type cytokines and foster CTL development. IL-12 is, therefore, critical in a series of immune-pathological conditions, such as viral infections and tumors [
47,
48]. On the other hand, IL-4 exists in elevated levels in sera of HCV infected patients than non-infected ones [
49]. It is known to down-regulate cell-mediated immune effector mechanisms important in the host defense against intracellular pathogens [
50]. IL-10 is a pleiotropic cytokine traditionally considered as immunosuppressive and anti-inflammatory, produced by many cell types, [
51,
52] which exerts its effects by inhibiting macrophage and DCs functions. In chronic HCV infection, patients have high serum levels of IL-10, associated with incomplete responses to INF therapy [
53].
RT-PCR analysis of immunized mice splenocytes m-RNA demonstrated an increase in mRNA expression of IL-12p40 and INF-γ transcripts in iDCs-core, and iDCs groups (Fig.
2), indicating a potent up-regulation in Th1 arm. While a down-regulation in mRNA expression in IL-10 transcripts was demonstrated at all groups that were immunized with different subsets of DCs with the lowest IL-10 mRNA expression in case of iDCs-core group (Fig.
2), indicating a down-regulation in Th2 arm. Protein expression of intrasplenic INF-γ, (the target protein) was high and in accordance with its mRNA expression (Fig.
3). This ascertains that the immune system is having a better struggle with the viral infection to prevent its persistency. On the other hand, protein expression of IL-12 was unexpectedly diminished. One mechanism that might account for the low intra-splenic IL-12 protein level was that it might be consumed by the cells [
54]. Alternatively, IL-12 has a half-life of only 12 h), [
55] or may have released out of the cell and functioned extracellularly. This postulate was assisted by the up-regulation of splenic IL-12p40 mRNA expression.
In the present study, the strongly reduced extracellular levels of blood NO of all immunized mice with different DCs subsets, with the lowest NO level in iDCs-core group, was indicative for a selective and persistent up-regulation of T cells INF-γ production. This finding was supported by Roozendaal et al. [
56] where NO was able to modulate the balance between the expression of Th1- and Th2-type cytokines through selective and persistent inhibition of expression of INF, shifting Th1/Th2 balance towards Th2 cytokines. It is worth noting that the main function of this vaccine was to counteract the effect of HCV infection of misbalancing the Th1/Th2 model.
CTL assay was performed to determine and quantitate the capacity of effector pre-induced cells (splenocytes of nDCs-core, iDCs-core groups individually), to eliminate target cells (EL4-core cells). The principle of this assay was to prime T cells to be antigen-specific through the immunization process and then, upon repeated exposure to a specific antigen, induce a rapid T cell expansion. The study carried out to determine the best number of co-culture between splenocytes and EL4 proved to be essential to ensure that EL4 and splenocytes were able to be cultivated together, and to determine the optimum effector:target cells ratio with the highest viability to use it for T lymphocyte suppression assay. CTL assay showed that splenocytes of iDCs-core group had a good T lymphocyte suppression capacity only on EL4-core cells (Fig.
4), indicating a specific T response to the core-pulsed cells. In this experiment, DCs have been successfully used as cellular adjuvants in mice to elicit protective T cell immunity against HCV core protein.
Anti-core humoral response
To determine if adequate levels of antibodies had been attained following vaccination, IgG antibodies recognizing HCV core protein were measured. Humoral response assessment revealed specific anti-core IgG antibodies only in iDCs-core group, with a remarkable high percentage against negative control (Fig.
5). This result reflects a significant B cells activation to produce specific antibodies against core protein. Interestingly, iDCs-core group that demonstrated the most significant humoral response also showed the highest cellular immune response.
In conclusion, the present study provides evidence for the role of BCE on stimulating splenic DCs proliferation, on both sides of DCs maturation and count. In addition, the basics for a novel vaccination strategy to treat HCV were manipulated, through analyzing the humoral and cellular immune response upon immunizing mice with different subsets of treated DCs. Therapeutic efficacy of BCE synergized core-pulsed DCs vaccination exhibited an interesting anti-core boosting effect against a tumor model expressing HCV core protein in an immunocompetent host (EL4-core cells). DCs are essential for T cells activation and since viral clearance in HCV infected patients is associated with a vigorous T cells response, a new type of HCV vaccine of a strong antigenicity in humoral and cellular immunities was proposed, based on ex-vivo stimulated HCV core protein-pulsed DCs and promoted by BCE treatment. This vaccine might have a therapeutic setting to potentially circumvent the diminished and down-regulated DCs function of HCV infected patients by giving the necessary maturation stimuli whether in vivo (BCE treatment) or in ex vivo (core protein transduction). As a therapeutic immunization approach, this vaccine may pertain new aspects compared to other vaccines, which are faced in HCV infected patients with down-regulated DCs function.
It is crucial that further studies should be carried out to reliably move this preclinical study to a clinical one. The ultimate goal in the near future is to conduct some research on the CD4 and CD8 T responses, and IL-28b monitoring, in order to full scheme the therapeutic influence of the proposed vaccine. Working on peripheral blood of HCV infected patients should be included. Finally, further clinical studies should be carried out on pegylated interferon non-responders HCV patients with low doses of vaccine tailored from each patient’s blood, for tracking its therapeutic effect.