Crosstalk between apoptosis and cytotoxic lymphocytes (CTLs) in the course of Lagovirus europaeus GI.1a infection in rabbits

Even though the rabbit disease caused by Lagovirus europaeus (L. europaeus) GI.1 or rabbit haemorrhagic disease virus (RHDV), now also referred to as acute hepatitis, was first recorded over 30 years ago (24), its mechanism of pathogenesis is still not well understood. It is assumed that the main pathogenic phenomenon of this virus is its affinity to the endothelium of blood vessels, which leads to disseminated intravascular coagulation (DIC) syndrome (1, 24) as a result of changes in the liver, including those manifested by apoptosis and necrosis of hepatocytes (3, 11, 22, 30, 34, 38, 42). Other important symptoms related to the pathogenesis of this disease caused by L. europaeus GI.1 (RHDV) are changes in animal immunity determined by the role of neutrophils, which are the first line of defence against this infection (19, 31, 40, 41). However, due to the replication of L. europaeus GI.1, (including GI.1a) and L. europaeus GI.2 viruses in liver cells, an important element in the pathogenesis of this disease is the apoptosis of hepatocytes (2, 11, 22) and kidney cells (5), in addition to apoptosis of lymphocytes (25, 32, 33, 38), granulocytes (31, 33), and monocytes (2, 12), which results in a change in the immune potential of infected rabbits (31). This was confirmed by the induction of anti-apoptotic factors, such as N-acetylcysteine (13), cardiotrophin (43), and melatonin (7, 42), which leads to a decrease in apoptosis and thus prolongs the life of rabbits infected with L. europaeus GI.1. It has also been shown that interleukin (IL)-10 prolongs the lives of rabbits infected with this virus (40, 41). Furthermore, an increased level of cytokines, such as tumour necrosis factor alpha (TNF-α), interferon alpha (IFN-α), and IL-1,6,8, in young rabbits infected with this virus results in an asymptomatic course of the disease (25). In contrast, in adult rabbits, increased levels of IL-10 and transforming growth factor beta (38) do not impact the lifespan. Nevertheless, it can be assumed that the process of apoptosis of the immune system cells is an important element of the pathogenesis of this infection because of the influence of anti-apoptotic factors on the course of rabbit infection with L. europaeus GI.1, which has also been confirmed in several virus infections (1, 36). Moreover, it is known that viruses replicate in the host organism, spread in organs and tissues, and seek a new organism to colonise; hence, the process of apoptosis in this chain of events may be an advantage or a disadvantage (1, 36).

The phenomenon of apoptosis as a defence strategy in viral infections has often been recorded, because during this process both viral and host proteins and nucleic acids are degraded (1, 16, 28, 36). The anti-apoptotic effect most often occurs as a result of the encoding proteins inactivating caspases, which are the executive proteins of this process (1, 16, 28, 36). Viral proteins blocking surface receptors involved in the transmission of the death signal, e.g. those of tumour necrosis factors and interferon gamma (IFN-γ), also play a significant role in inhibiting apoptosis (1, 16, 28, 36). As a result of this action, viruses enter the host cell, inhibit apoptosis, and replicate and spread to other cells (16). The inducing effect of viruses on the apoptosis process has also been described, although it usually concerns apoptosis along the mitochondrial pathway (1, 7, 28, 45). In such a case, the virus enters the cell and induces the apoptosis process, which leads to the cell’s death (10, 16, 29). It has also been reported (16, 29) that many RNA viruses, such as L. europaeus GI.1 produce a large number of virions by rapid replication before the apoptosis process and possible immune response are triggered, and this may be their path in pathogenic activity. Apoptosis has been shown to occur in viral infections (1, 16, 28, 36) in many immune cells, both in peripheral blood (2, 12, 25, 32, 33, 38) and in organs (2, 5, 11, 22), thereby depleting the number of such cells, including CTLs with CD8+ receptor (4, 15).

In L. europaeus GI.1 infection, increased activity of CTLs was noted in infections of more than 30 strains of this virus (18) and five strains specifically of L. europaeus GI.1a (31). Infections with two strains of L. europaeus GI.2 also potentiated this activity (14, 27). It has been proven that the process of apoptosis occurs more efficiently when the pseudoreceptor (granzyme/ perforin) pathway is additionally involved. In this case, in the target cell’s membrane a channel is formed by perforin, and the cytoplasmic grains, which contain granzymes and initiate apoptosis, enter the cell (4). Therefore, it can be assumed that the number of CD8+ T lymphocytes in the peripheral blood in rabbits infected with L. europaeus GI.1 is associated with the process of apoptosis, which would be pivotal in the pathogenesis of RHD in rabbits.

Studies on the apoptosis of granulocytes and peripheral blood lymphocytes in rabbits infected with this virus showed that this process was involved in both types of immune cells, but that it occurred more intensively in peripheral blood lymphocytes and, regardless of the type of cell, it took place as soon as 4–8 h post inoculation (p.i.) (31, 32, 33). Therefore, the aim of the current research was to assess the crosstalk between the apoptosis of peripheral blood lymphocytes and CTLs in rabbits infected with six L. europaeus GI.1a viruses, and to record the clinical status and mortality of the tested animals.

Animal trial. The research was carried out on 120 Polish hybrid rabbits from a licensed breeding farm, of both sexes, weighing 3.2–4.2 kg (35). During the experiment, the animals were kept under constant veterinary supervision in the vivarium of the Department of Microbiology and the Department of Immunology, in the Institute of Biology at the University of Szczecin. The zootechnical parameters were consistent with the national standards developed in accordance with the European Union Directive in terms of temperature, humidity, lighting and the size of animal cages (13). The rabbits were divided into an experimental group of 60 animals and a control group of 60. After being transported to the vivarium, the subjects were allowed a two-week adaptation period. The animals were provided with complete feed for rabbits and had unlimited access to water. The study was approved by the Local Ethics Committee in Szczecin, Poland (approval no. 1/2006). All procedures involving animals were carried out in accordance with the Polish legislation on animal welfare.

Viruses used in the study. Rabbits were administered 6 L. europaeus GI.1a viruses derived from rabbits which died of naturally acquired infection. The strains, obtained in 1996–2003, are designated 9905, Pv97, Hartmannsdorf, Triptis, Vt97, and 72V/2003 (Table 1). The viruses are systematically noted as L. europaeus GI.1a according to the information from the donating institutions and the NCBI nucleotide database. The rabbits were infected with these strains in the form of 1 mL of a 20% liver homogenate, purified by centrifugation at 3,000 rpm, addition of 10% chloroform for 60 min and another centrifugation, and suspended in glycerol in a 1 : 1 ratio (31). All antigens prepared from the six viruses of L. europaeus GI.1a had the same number of viral particles determined by the density in caesium chloride of 1.34 g/dm³ (31). The animals of the infected groups (10 for each virus) were lower limb intramuscularly administered the L. europaeus G1.1a antigen suspended in 1 mL of glycerol, and, in the same manner, the rabbits of the control groups (also 10 for each virus) received 1 mL of glycerol as a placebo.

Experiment scheme. Blood was drawn for anticoagulant and collected for the tests from rabbits of the infected and control groups from the marginal vein of the ear at 0 h, i.e. before the inoculation, and then at 4, 8, 12, 24, and 36 h p.i. All animals died as a result of viral infection between 24 and 36 h p.i. During this time, clinical signs were recorded, as was the percentage of animal mortality, and every 24 h, always at 8 a.m. on each day of the experiment, the conditions in the vivarium were monitored in terms of temperature and air humidity.

Apoptosis measurements. Peripheral blood lymphocyte apoptosis was measured using the ApoFluor Green Caspase reagent kit (MP Biomedicals, Santa Ana, CA, USA) on a FACScan flow cytometer with CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA), and was assessed by recording the activity of the total caspases 1, 3, 4, 5, 6, 7, 8, and 9. The result was given as the percentage of apoptotic lymphocytes (32). Heparinised blood was centrifuged for 5 min at 400 × g, then 300 μL of the suspension was transferred to Eppendorf tubes and 10 μL of the ApoFluor Green fluorescent dye was added, mixed, and incubated for 60 min at 37°C in 5% CO₂. Then 2 mL of washing buffer was added, and the solution centrifuged for 5 min at 400 × g at room temperature. After removing the supernatant, the centrifugation procedure was repeated, after which 400 μL of washing buffer and 2 μL of propidium iodide (PI) were added to the pellets of cells stained with ApoFluor Green. The prepared test tubes were incubated on ice for 10 min and analysed on the flow cytometer. With the use of the two dyes ApoFluor Green and PI we distinguished apoptotic and necrotic cells. The results were interpreted by counting the percentage of apoptotic granulocytes and lymphocytes in the total cell pool as the flow cytometer registered the lymphocytes as Apo (+) PI (−) cells stained with ApoFluor Green dye and unstained with PI or Apo (+) PI (+) stained with both ApoFluor Green and PI, and these cells were treated as apoptotic.

CTLs (CD8+ lymphocytes) flow cell analysis. The percentage of T cells with the CD8+ receptor was cytometrically assessed on FACScan flow cytometer with FACSDiva software (Becton Dickinson) using mouse anti-rabbit monoclonal antibodies (AbD Serotec, now Bio-Rad, Hercules, CA, USA) (31). Test samples were incubated for 45 min on ice, and Cell Wash (BD Biosciences, Franklin Lakes, NJ, USA) was used three times with 200 × g centrifugation. A 10 μL aliquot of rabbit antibody labelled against mouse IgG with fluorescein isothiocyanate was added. After repeating the washing procedure three times in Cell Wash, 2,000 μL of erythrocyte removal lysis fluid (BD FACS Lysing Solution; BD Biosciences) was added to the samples and the measurement was performed after 10 min of incubation in the dark at room temperature. The results were processed with Student’s t-test at the significance level set at P < 0.05 with Statistica 6.0 (StatSoft, Tulsa, OK, USA).

The activation of the process of apoptosis in peripheral blood lymphocytes was observed in infections with all six L. europaeus GI.1a strains (Table 1), starting as early as 4 h after infection and lasting up to 36 h p.i. (Figs 1 and 2). The percentages of the full pool of cells which were apoptotic peripheral blood lymphocytes ranged from 29.08% to 57.66%, with an increasing trend between 12 and 24 h p.i. (Fig. 1), and the percentages of CTLs in relation to the entire peripheral blood pool value ranged between 5.90% and 23.74% (Fig. 2). The trend of these values over time was inverse to the size of the total pool of apoptotic lymphocytes, because the percentage of CTLs in the total blood pool decreased from 8 to 36 h p.i. (Figs 1 and 2). Symptoms of the disease occurred at 24–36 h p.i. and were specific and typical for the disease, but different percentages of mortality by strain were observed in the animals at 36 h p.i.: 100% for Hartmannsdorf, 72V/2003 Triptis and Pv97 infections; 90% for 9905 RHDVa infections; and 30% for Vt97 infections.

Fig. 1

Fig. 2

The phenomenon of apoptosis in peripheral blood cells (lymphocytes and granulocytes) in rabbits infected with L. europaeus GI.1 was described for three non-haemagglutinating virus strains (Rainham, Frankfurt and Asturias), where the process was induced from 4 to 36 h p.i. and was more intense in lymphocytes than in granulocytes (32). However, in the case of haemagglutinating GI.1 strains (24V/89, 1447V/96, 01-04, 237/04, V-412 and 05-01) (33), apoptosis started later, at 8 or even 12 h p.i., and was more intense in peripheral blood granulocytes. In contrast to the case of non-hemagglutinating L. europaeus GI.1, the intensity was observed in lymphocytes (33). The results differed in the present study in terms of the intensity of the apoptosis process. In the present study’s rabbits infected with Lagovirus europaeus GI.1a, comparably to those infected with Lagovirus europaeus GI.1 in the cited research, this process occurred during the period from 4 to 36 h p.i. and was more intense in lymphocytes.

In contrast, the analysis of the full picture with L. europaeus GI.1 and GI.1a, which differ in terms of haemagglutinating capacity, showed that the percentage of CD8+ T cells in blood samples from non-haemagglutinating L. europaeus GI.1 virus–infected rabbits was characterised by an increase in the Rainham strain infections 24 h p.i., a decrease in the Asturias strain infections at 4 h p.i., and no changes in the Frankfurt strain infections (31). A similar picture emerged for the infections with haemagglutinating L. europaeus GI.1 (19), in which increases were noted (mainly at 8–12 h p.i. for strains V-412, 24V/89 and V-561, and a decrease at a more advanced time of infection, i.e. 24–36 h p.i. was manifested for strains 01-04, 237/04, 05-01, KGM and PD.

CTLs are essential to the elimination for virus-infected cells because they killed target cells, mainly via perforin-mediated granzyme delivery following exocytosis of cytotoxic granules (8, 9, 20, 23). Lytic molecules such as perforin, granzymes, and granulysin could also participate in this process, which affected apoptosis (4, 44, 45). It has been shown that the amount of perforin also correlated with the cytotoxic ability of lymphocytes because the expression of PRF1 increased as the cytotoxicity of T lymphocytes did (45).

The second pathway is the activation of the Fas ligand–mediated coupling of the target cell Fas death receptors (4). Immune synapse formation between CTLs and target cells triggers the rapid orchestration of the granule exocytosis pathway (9, 20). At the immune synapse, granzymes enter the target cells following membrane perturbation by perforin (9, 20). Granzymes are serine proteases, capable of cleaving numerous substrates in the cytoplasm and nucleus required for induction of apoptosis. Direct cleavage and subsequent activation of caspases, mainly of caspase 3 by granzyme B, is thought to be a critical first step in CTL-induced apoptosis (6, 45). Granzymes and caspases may also have a common molecular target, leading to apoptosis (17, 44, 45) In particular, granzyme A most effectively “cleans up” after viral infection; hence, the amount of GrA increases significantly in the acute phase of Epstein–Barr virus and human immunodeficiency virus (HIV) infection, in addition to in respiratory syncytial virus and Dengue virus infection (44).

In chronic infections in humans with HIV and hepatitis C infections, and in these in mice with lymphocytic choriomeningitis virus and Friend retrovirus, it was shown that the CTL causing these infections became exhausted (8, 23). Terminally exhausted T (T_EX) cells are characterised by an impaired ability to proliferate and produce cytokines, e.g. IL-2, TNF and IFN-γ, and also show increased expression of receptors suppressing the immune response, i.e. programmed cell death 1 (PD-1) and programmed target death ligand 1 (PD-L1). This leads to increased apoptosis and, hence, viral escape from immune surveillance (23). Moreover, the presence of precursor exhausted T (T_PEX) cells ) was recorded prior to the appearance of T_EX and with a phenotype similar to memory and follicular T cells (21, 23).

In acute viral infections caused by retroviruses (8) and influenza virus infection (26), there is a lack of expression of receptors suppressing immune responses, i.e. PD-1 and PD-L1 immunological checkpoints, which leads to increased apoptosis. In addition, in vaccinia, influenza and rabies virus infections in mice, and hepatitis A, B, and C viruses in humans, increased expression of PD-1 was recorded (21). Moreover, during infection with acute viruses, T lymphocytes react precisely through the expression of PD-1, and, in addition, PD-L1 expression in various cells increases, either directly through pattern recognition receptor activity or indirectly through the release of inflammatory cytokines (21).

The changes in rabbits infected with six L. europaeus GI.1a viruses showed that during this infection, apoptosis of peripheral blood lymphocytes was triggered in the early stage of infection, i.e. at 4 h, and lasted up to 36 h p.i. In addition, as the number of apoptotic lymphocytes increased, the number of CTLs decreased. The greater intensity of apoptosis in lymphocytes and simultaneous decrease in the number of CTLs in rabbits infected with L. europaeus GI.1a indicate CTL-induced apoptosis, which is a novel element in the pathogenesis of the disease in rabbits. This appears to be due to the triggering of programmed CTL-induced cell death because of the change of the CTLs phenotype to T_EX. This is likely to significantly influence the course of viral infection in rabbits caused by L. europaeus GI.1a, resulting in a decreased response by CTLs. Such an effect is also registered clinically, in 90–100% mortality of the infected animals up to 36 h p.i.

The immunological difference between L. europaeus GI.1a and GI.1 confirmed in previous studies (31) was further highlighted in the current study, in which a negative correlation was found between apoptosis of lymphocytes and the number of CTLs. This result is the first proof of virus-induced CTL apoptosis in L. europaeus GI.1a infection. Further investigations are needed to determine whether activation of caspase 3 might be a critical point in this type of CTL-induced apoptosis, and if PD-1 and PD-L1 are immune checkpoints in more infections than only L. europaeus GI.1a.