Effects of lactoferrin on viral replication in B cell lines corresponding to various stages of B cell differentiation

Introduction

B cells are a major target of viral infections, leading to the development of neoplastic and autoimmune diseases [1, 2]. Viruses access cell targets via a variety of receptors including heparin sulphate [3], DC-SIGN molecule [4], CD150 [5], CD21 [6], co-receptors [7], and other, nonspecific routes [8].
The susceptibility of B cells to viruses, at defined stages of development, may vary and lead to various consequences. Transformation of immature B lymphocytes with Abelson murine leukaemia virus, expressing the v-abl oncogene, induced pre-B cell lymphomas and kappa light rearrangement [9], but the target cells belonged to different developmental stages. On the other hand, the ABL-MYC virus, expressing both v-abl and c-myc, produced only plasmacytomas [10]. In another murine model, pim 1 and myc proto-oncogenes transformed precursor but not mature B lymphocytes [11]. Epstein-Barr virus infection of immature human B cells resulted in a switch of complement receptors [12]. Although gamma herpes viruses are detected in bone marrow pro-pre B cells and immature B cells during early and long-term latency, their possible access to the mature B cell compartment is also suggested [13]. Depletion of bone marrow during influenza infection in mice, associated probably with apoptosis, uses also indirect mechanisms such as involvement of TNF- and lymphotoxin  [14].
Lactoferrin (LF) is one of the proteins involved in iron metabolism and is contained in excretory fluids of mammals and secondary granules of neutrophils [15]. The protein is known for its protective properties against all classes of pathogens, including viruses [16]. The antiviral actions of LF include neutralisation of virus [17], competition with cell receptors for viruses [18], and interference with viral replication by various mechanisms [19]. Recent studies showed that iron-saturated LF was more efficient in inhibition of virus replication than iron-depleted LF [20]. In addition, LF encapsulated in liposomes exerted a stronger antiviral action than free LF [21].
The present investigations were performed on 3 B-cell lines differing in their differentiation stage. WEHI-231 is a mouse B-cell lymphoma with an immature cell phenotype, highly susceptible to apoptosis upon crosslinking of IgM surface receptors and by viruses that can overcome a survival signal by CD40 receptor ligation on these cells [22]. A-20 mature B-cell line is highly susceptible to viral infection, so this feature is of value in exploring oncolytic activities of viruses in in vivo models [23]. In addition to WEHI-231 and A-20 B cell lines, 7TD1, IL-6-dependent plasmacytoma cells [24] were included in the study because data on susceptibility of plasmacytoma cells to viruses are lacking. Encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and human herpes virus type 1 (HSV-1) were applied for cell infection. Bovine (bLF) and recombinant mouse (rmLF) lactoferrins were used to evaluate antiviral protection measured by a cytopathic effect.

Aim of the research

The aim of this investigation was to evaluate the susceptibility to viral infection of B cell lines, representing different stages of B cell differentiation, and to determine the protective effects of mouse and bovine lactoferrins on viral replication in these cells.

Material and methods

Cell lines

All cell lines and viruses were derived from the cell bank of the Institute of Immunology and Experimental Therapy, Wrocław, Poland. WEHI-231 cells – a mouse B cell lymphoma (ATCC CRL 1702), A-20 cells – a murine B cell lymphoma (ATCC TIB-208), 7TD1 cells –murine plasmacytoma (ATCC CRL-1851), L929 cells – a murine fibroblast-like cell line (ATCC CCL 1), and A549 cells – human adenocarcinoma lung cells (ATCC CCL185) were used and were maintained according to the manufacturer’ instructions. L929 cells were applied as a reference line for titrated EMCV and VSV viruses, and the viral cytopathic effect (CPE) is shown in Figures 1 A–E. The CPE of the HSV-1 in the reference A549 cells is shown in Figures 2 A–C.

The viruses

EMCV – encephalomyocarditis virus (ATCC VR-129BTM); Picornaviridae; non-enveloped RNA virus was propagated in L929 cells.
VSV – vesicular stomatitis virus (ATCC VR-1238); Indiana strain, Rhabdoviridae, enveloped RNA virus was propagated in L929 cells.
HSV-1 – human herpes virus type 1 (ATCC VR-539™); McIntyre strain, Herpesviridae, enveloped DNA virus was propagated in A549 cells.
The titres of the viruses were expressed in reference to the value of TCID50 (tissue culture infectious dose), based on the cytopathic effect caused by this virus in about 50% of infected cells. EMCV and VSV viruses were used at the dose of 101 TCID50/ml, and HSV-1 at the dose of 104 TCID50/ml.

Infection of cell lines and treatment with lactoferrins

WEHI-231, A-20, and 7TD1 (1 × 105 cells/ml) were infected with a dose of 101 TCID50/ml of EMCV or VSV and 104 TCID50/ml of HSV-1. Following a 45-min incubation at 37°C, the medium was removed and the LFs were added (50, 25, 12, or 6 µg/ml). Non-treated and cells treated with viruses served as controls. Following 24 h, 48 h, and 96 h incubation, the supernatants were collected and stored at –80°C until virus titration using a standard TCID50 method with a two-fold serial dilution. Briefly, the supernatants were diluted and adsorbed to L929 or A549 cells at a density of 1 × 105 cells/ml in 96-well culture plates. Following 24 h, 48 h, and 96 h of incubation at 37°C the viral cytopathic effect (CPE) was analysed under a microscope (Figures 1 A–E and 2 A–C). The TCID50 was calculated by determining the end-point dilution of the virus, where 50% of the infected cells displayed CPE. The antiviral activity was determined by comparing the logarithmic reduction factor (log 10) of the viral titre with the control. The results obtained from 3 independent experiments are presented as log TCID50 mean values ± standard deviation (SD). When considering significance of the antiviral activity of LF, we refer to a commonly accepted definition that ≥ 4 Log 10 reduction of virus titre is typical for the action of chemical disinfectants, resulting in 99.99% elimination of the virus.
The photographs show some typical morphological changes in cells caused by the virus, such as loss of monolayer integrity, lysis, granulation, and vacuolisation in the cytoplasm. These pathological changes were absent upon addition of bLF at a concentration of 50 µg/ml (Figures 1 A–E and 2 A–C).

Results

A-20 cells were most prone to viral infection. The titre of EMCV in these cells after 24 h, 48 h, and 96 h was 8-log, of HSV-1: 7; 7.8 and 7.7-log, respectively, and that of VSV: 7.5; 7.7 and 7.7-log in the respective time points (Figure 3 A).
The titres of EMCV in WEHI-231 were 6.2; 7.5 and 8-log at respective time intervals. The titres of HSV-1, in turn, attained 3.7-log after 24 h and 7.5-log after 48 h and 96 h. VSV virus, at these time intervals reached titres of 5.2; 5.2 and 5.7-log (Figure 3 B).
The levels of viral replication in 7TD1 cells were much lower for EMCV after 24 h and 48 h equal to 5.6-log and 6.3-log after 96 h (Figure 3 C). The HSV-1 titres at respective time points attained 2.7-log (24 h, 48 h) and 3.0-log at 96 h. VSV titre was 2.6; 4.3 and 5.3-log at measured incubation times.
Both LFs lowered the viral titres, with the highest inhibitory effect in A-20 cells. In these cells bLF (50 µg/ml) reduced the HSV-1 level from 7.8-log to 2.5-log, at 25 µg/ml from 7.8-log to 3.0-log, and at 12.5 µg/ml to 4.5-log after 48-h incubation. bLF significantly decreased HSV-1 in a dose-dependent manner. Also, a significant reduction of HSV-1 titre by rmLF occurred after 24 h, from 7.0-log to 2.3-log at 50 µg/ml (Figure 4 A).
The inhibitory effects of LFs on virus replication in WEHI-231 and 7TD1 cells was also registered, albeit at a lower efficacy, where bLF exhibited a better potency than rmLF (Figures 4 B, C).

Discussion

In this investigation the lactoferrins differentially inhibited multiplication of EMCV when applied after viral infection. Hence, these actions consisted of interference of LFs with the viral multiplication and were not dependent on interaction of LFs with surface cell receptors for viruses. Recently, we demonstrated significant effects of both types of LFs on inhibition of EMCV replication in B-cell-enriched splenocyte populations in young versus old mice [25]. Such splenocytes may be considered as an enriched population of mature B cells, so these results with resident cells correlate with the present study on A-20 cell line. Our results also confirmed other findings that A-20 cells are highly susceptible to viral infection [23]. In the present work bLF was much more effective in inhibiting viral replication when applied in culture after the infection, as compared to a pretreatment of the cells with LF. Similar results were obtained with resident splenic cells [25]. Because the main common cell receptor for LF and viruses is heparin sulphate [3, 17], other routes of viral entry, not interfered by LF, may account for this difference in LF antiviral actions.
The inhibitory effects of LF on virus replication and the susceptibility of A-20 cells to infection varied, depending on the type of virus. In the evaluation of the antiviral activity of LF we must consider a commonly accepted measure that ≥ 4 Log 10 reduction of virus titre is virucidal for chemical disinfectants because it leads to 99.99% reduction of viral titre [26]. A much deeper inhibition of viral replication was registered with HSV-1 than with VSV virus. This discrepancy could be due to different cell receptors used by these viruses, where VSV uses angiotensin-converting enzyme 2 (ACE2) [27] and HSV-1 – heparan sulphate [28] efficiently blocked by LF, as shown by others [17]. Although the target cells were washed after preincubation with LF, such a procedure cannot reverse firm attachment of LFs with cellular heparin sulphate receptors and subsequent block of virus entry.
In turn, the low susceptibility of 7TD1 cell to the virus probably results from a lack of most relevant receptors on these cells in the terminal stage of B-cell differentiation. Thus, in theory, antibody-producing plasma cells may be less susceptible to viral infection. On the other hand, at this stage of investigation, it is hard to explain why homologous rmLF was effective in WEHI-231 but not in A-20 cell line.
Considering the ability of lactoferrin to enter cells, the protein may affect viral RNA. In fact, bLF significantly decreased the content of intracellular viral RNA in bovine kidney MDBK cells infected with enterovirus E [28, 29]. However, the effective concentration of LFs, leading to reduction of the cytopathic effect in our model (50 µg/ml), was many times lower than reported by these authors (1 mg/ml). This difference could be due to another experimental model used in their study.

Conclusions

The degree of infection was dependent on a stage of B-cell maturation. Mature B cells (A-20) were most susceptible to viral infection. Lactoferrins suppressed multiplication of 3 types of viruses to various degrees, and bLF was more effective than rmLF. We postulate that lactoferrin has a potential therapeutic utility in viral infections of B cells, applied together with classical antiviral drugs.

Funding

This study was supported by statuary grant no. 4/2014 from the Polish Ministry of Science and Education.

Ethical approval

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

References