REACTIVE OXYGEN SPECIES PRODUCTION IS SUPPRESSED IN

4 Ergebnisse

4.2 REACTIVE OXYGEN SPECIES PRODUCTION IS SUPPRESSED IN

VIRULENT STRAIN

Maren Martin¹, Regina Carlson¹, Wolfgang Baumgärtner², Andrea Tipold¹, and Veronika M. Stein¹

1Department of Small Animal Medicine and Surgery, ²Department of Pathology, University of Veterinary Medicine, Hannover, Germany

Corresponding author: Maren Martin

Department of Small Animal Medicine and Surgery University of Veterinary Medicine Hannover

Buenteweg 9 D-30559 Hannover Germany

Tel. 0049-511-953-6200 Fax 0049-511-953-6204

E-Mail: Maren.Martin@tiho-hannover.de

63 ABSTRACT

Canine distemper virus (CDV) infection induces immunosuppression. Microglia cells are shown to be activated and generate reactive oxygen species (ROS) which might represent one mechanism of virus clearance leading simultaneously to demyelination in the central nervous system (CNS). CDV infection causes recruitment of peripheral blood monocytes to the CNS to become tissue macrophages. In this environment they show effector functions such as phagocytosis and ROS generation which makes them indistinguishable from microglia. However, their contributing role to the pathogenesis of virus clearance and demyelination is so far not defined. Therefore, monocytes were isolated with density gradient centrifugation from 10 mL of whole blood from 10 healthy Beagle dogs and infected with either an attenuated CDV vaccination strain (Onderstepoort, OND) or a virulent CDV strain (R252). The expression of CD14 and the ROS generation were measured directly and 3 hours post infection (p.i.) by flow cytometry. Non-infected monocytes were used as negative control (ctr). Both CDV strains were capable to infect the isolated monocytes but the percentages and the virus load were significantly lower with the OND (mv = 32.5%) compared to the R252 strain (mv = 61.2%). The expression of CD14 was approximately 20% higher in the infected monocytes (OND mv = 71.6%, R252 mv = 68.9%) compared to the ctr (mv = 50.3%). Furthermore, the CD14 expression intensity was significantly up-regulated in CDV-infected monocytes compared to the ctr (3 hours p.i., OND: 4.9fold, R252: 4.7fold). CDV infection of peripheral blood monocytes results in a distinct ROS generation, but interestingly, CDV-infected monocytes synthesized significantly less ROS than the non-infected ctr. Intriguingly, the virulent R252 strain generated less ROS than the attenuated OND which might represent a possible mechanism for the virulent strain to escape the immune surveillance and pave the way to enter the CNS which can result in virus persistence.

Keywords: monocytes, canine distemper virus infection (CDV), pathogenesis, reactive oxygen species (ROS), CD14, demyelination

64 INTRODUCTION

Canine distemper virus (CDV) belongs to the genus morbillivirus and infects carnivores worldwide (Deem et al., 2000; Pringle, 1999). Primary virus replication is seen in the cells of the immune system which leads to apoptosis and therefore to massive immunosuppression and leucopenia (Kumagai et al., 2004; McCullough et al., 1974; Schobesberger et al., 2005). In a second step the virus spreads via haematogenous pathways to the gastrointestinal tract, the urogenital tract and to the respiratory system (Appel, 1969; Okita et al., 1997). The virus enters the central nervous system (CNS) mainly haematogenously as free virus particles or bound to thrombocytes and lymphocytes (Krakowka, 1989; Krakowka et al., 1987). Other pathways for entering the CNS are through endothelial cells of the meninges (Bäumgartner et al., 1989) or the liquor cerebrospinalis (Vandevelde et al., 1985).

Once in the CNS, the virus induces activation of the microglia cells which will change their functionality and immunophenotype (Stein et al., 2004b), they release reactive oxygen species (ROS), start phagocytosis and act as antigen presenting cells through expression of MHC and CD1c (Stein et al., 2004b). Although, ROS is essential for defence and repair mechanisms (Gilgun-Sherki et al., 2004) it can also be cytotoxic once released in a vulnerable environment such as the CNS. It therefore causes degeneration of oligodendrocytes as innocent bystanders (Stein et al., 2004b) resulting in damage to the myelin sheath (Griot et al., 1990). CDV infection recruits peripheral blood monocytes to migrate into the CNS and become macrophages to support the immune response of microglia (Griot-Wenk et al., 1991a, b; Wisniewski et al., 1972).

Viral infections in general lead to activation of monocytes through a CD14/TLR4-dependent pathway associated with pro-inflammatory cytokine production such as interleukin (IL-) 1β, IL-6, and tumor necrosis factor (TNF)-α to further activate the innate immunity (Rolland et al., 2006). It has been shown that monocytes of in vivo CDV-infected dogs present an increased expression intensity of the surface molecules CD1c, B7-2, MHC I, and CD11b, all of which play an important role in the host´s immune response (Stein et al., 2008). The percentage of CD14-positive monocytes was generally increased in the acute phase of the infection. However, in the dogs with severe neurological signs and demyelinating lesions in the CNS the CD14 expression was about 30% lower compared to monocytes of dogs with only

mild clinical signs without histopathological lesions in the CNS and of the non-infected control group. Therefore, the percentage of CD14-expressing monocytes reflects the activation state of the innate immune system (Stein et al., 2008) and leads to the assumption that CDV infection of the CNS with a virulent strain results in immunosuppression and reduced virus clearance.

The aim of our study was to prove the hypothesis that CDV-infected monocytes are capable to generate ROS. It should be evaluated to what extent the possible ROS generation can lead to systemic cell damage and after recruitment to the CNS may contribute to immunosuppression and virus clearance in the acute phase of CDV infection. Furthermore the ability to generate ROS was compared between two CDV strains of different virulence.

MATERIAL AND METHODS Animals

Whole blood samples of 10 mL of ten healthy one to six-years-old Beagle dogs were taken from the V. cephalica antebrachii or V. saphena lateralis by sterile venipuncture into heparinised tubes. The dogs were examined regularly and results of general examination as well as total blood count were unremarkable. All animal experiments were performed according to national regulations for animal welfare (animal experiment number 33.9-42502-05-12A275).

Antibodies

R-Phycoerythrine (PE)-conjugated primary monoclonal antibody (mAbs) against the cell surface marker CD14 (Clone TÜK4, DakoCytomation, Glostrup, Denmark) was used to identify the isolated monocytes together with their morphological characteristics. R-Phycoerythrine conjugated mouse immunoglobulin G2a (Clone PPV-04, Immunotools, Friesoythe, Germany) was used for the isotype control.

The murine mAb D110 from harvesting cell culture supernatant (Bollo et al., 1986) was used for detection of the CDV nucleocapsid protein. The antibody binds to the nucleocapsid protein which is resistant to tissue fixation and embedding procedures.

Non-specific bindings were blocked using human normal immunoglobulin G (IgG) (Human TrueStain FcX, BioLegend Inc., San Diego, USA) following the manufacturer’s instructions. The labelled cells were evaluated by flow cytometry (FACS) analysis.

Virus strains

The virulent CDV-strain R252 (R252, kindly provided by Dr. S. Krakowka) and the attenuated vaccine strain Onderstepoort (OND, kindly provided by Dr. von Messling).

Both viruses were cultivated in vero cells in a pathogen-free environment and stored at -80 °C.

Isolation of peripheral blood mononuclear cells (PBMCs)

Immediately following sampling, density gradient centrifugation with Histopaque (density 1.119, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and Pancoll (density 1.077; PAN BIOTECH GmbH, Aidenbach, Germany) (Somberg et al., 1992;

Wunderli and Felsburg, 1989) was performed at 700 x g for 30 minutes at room temperature with the blood diluted in phosphate buffered saline (ratio 1:1; PBS).

Mononuclear cells were collected from the interphase between plasma and Pancoll and washed three times with PBS. After the last washing step the cells were infected with either the attenuated OND or the virulent R252 CDV strain, both at 0.1 multiplicity of infection (MOI). The negative control (ctr) was incubated with Hanks´ buffered saline solution (HBBS, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The cells (ctr, Ond-infected, R252-infected) were evaluated by flow cytometry for their CD14 expression, CDV content, and ROS generation after incubation for one hour (directly post infection, p.i.) and for additional three hours (37°C and a carbon dioxide level of 5%; 3 h p.i.). These points of measurement were chosen because preliminary testing showed that cultivation past three hours resulted in a decrease of cell viability. Before the FACS measurement, the cells were washed three times with HBSS, centrifuged at 170 x g for ten minutes each at room temperature.

67 Flow cytometry

Analysis of the flow cytometry data was performed with the Cell-Quest^-Software (Becton and Dickinson GmbH, Heidelberg, Germany). Monocyte and lymphocyte populations were differentiated by their size (forward scatter, FSC) and complexity (side scatter, SSC).

Detection of the surface molecule CD14

Membrane immunofluorescence for detection of CD14 on monocytes was performed as previously described (Stein et al., 2008). Fc-receptor blockade was attained using human IgG. Primary labelled PE-conjugated CD14 mAb (dilution of 1:8), the isotype (dilution of 1:5) and the negative control (HBBS) were incubated for 20 minutes at 4°C. After washing once with HBBS and centrifugation at 300 x g for ten minutes at 10°C the cells were resuspended in FACS flow solution (Becton and Dickinson GmbH, Heidelberg, Germany) with TO-PRO^-3 (dilution of 1:1.000, Molecular probes^, Life Technologies GmbH, Darmstadt, Germany) to assess cell viability and measured directly using a FACSCalibur™ (Becton and Dickinson GmbH, Heidelberg, Germany). Each dog was evaluated separately by flow cytometry to reveal percentages of CD14-expressing monocytes as well as CD14 expression intensities (mfi; measured by means of fluorescent channel numbers) for ctr, OND, and R252 at the two described time points (directly p.i. and 3 h p.i.). For each sample 10⁴ live-gated events were analysed. The analysis gate of the negative control was adjusted not to exceed a 2% cut-off of positive staining.

Detection of CDV infection of monocytes

Intracellular detection of CDV in monocytes was performed as previously described (Cherpillod et al., 2000). After fixation with 4% paraformaldehyde the monocytes were incubated for 20 minutes at 4°C and washed twice with PBS and centrifuged at 250 x g for ten minutes at 10 °C. The cell membrane was permeabilized with 150 µl of 0.03 % PBS-Saponin-solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) followed by Fc-receptor blockade using human IgG. 3.125 µL of the

primary antibody D110 (in a dilution of 1:16) was added and incubated for 30 min at 4°C. After two washing steps with 0.03% PBS-Saponin-solution and centrifugation at 250 x g for five minutes at 10 °C secondary goat-anti-mouse PE-conjugated IgG antibody (in a dilution of 1:50) was added to all samples and incubated again for 30 minutes at 4 °C. Following two last washing steps with 0.03% PBS-Saponin-solution and centrifugation at 250 x g for five minutes at 10 °C, the cell pellet was resuspended in FACSFlow solution and measured immediately by flow cytometry using FACSCalibur™. For each sample 10⁴ live-gated events were analysed and using the Cell-Quest^-Software the percentage of CDV-positive monocytes and the virusload (measured by means of fluorescent channel numbers; mfi) where assessed for ctr, OND-, and R252-infected cells at the two time points (directly p.i. and 3 h p.i.) The negative control was adjusted not to exceed a 2% cut-off of positive staining in the analysis gate.

Detection of ROS production in monocytes

Detection of ROS production of monocytes was performed as previously described for microglia cells (Stein et al., 2004b). For evaluation of the ROS production of infected monocytes the cells were triggered with phorbol myristate acetate (PMA, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) whereas the control received PBS. The test was performed in duplicates and mean values were assessed. 90 µL of cell suspension was filled into each tube and incubated for 15 minutes at 37°C with a carbon dioxide level of 5%. Either 10 µL PMA (diluted in dimethylsulfoxid to a concentration of 1 mmol and further diluted in PBS to a final concentration of 100 nmol) or 10 µL PBS were added and the cells again incubated for 15 minutes at 37°C. Following incubation, 20 µL of the non-fluorescent dihydrorhodamin 123 (DHR; 1.5 mg/mL diluted in dimethylsulfoxid and further diluted in PBS to a concentration of 15 µg/mL; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was added, followed by another incubation step. Cells were then placed on ice for 15 minutes, FACS flow solution with TO-PRO^-3 was added, and finally the cells were measured by flow cytometry using FACSCalibur™. For each sample 10⁴ live-gated events were analysed. The percentage of ROS generating monocytes and ROS generation intensity measured by means of fluorescent channel numbers) were

assessed with the Cell-Quest^-Software for the ctr, OND- and R252-infected cells at the two time points (directly p.i. and 3 h p.i.) seen as a shift in FL-1. The negative control received PBS instead of PMA and DHR.

Statistics

Statistics were performed using SAS^ version 9.2. (SAS Institute Heidelberg, Germany). Spearman´s rank correlation coefficient was calculated to evaluate any correlation between the different features of the infected monocytes; a value of p < 0.05 was considered significant. A one-way variance analysis as well as a two-way variance analysis was performed using the global F-test to compare the results of the ctr, OND-, and R252-infected cells and the two time-points of testing (p.i. and 3 h p.i.). The f-distribution was considered significant if f < 0.05. To evaluate the details of that analysis a Student’s t-test was performed, where p < 0.05 was considered significant.

RESULTS

Immunophenotypical characterization of monocytes during CDV infection CDV infected monocytes (OND and R252) showed a significantly up-regulated expression and expression intensity of CD14. The percentages of CD14-expressing monocytes was approximately 20% higher compared to the non-infected control directly p.i. (ctr: mv = 50.3%, OND: mv = 71.6%; R252: mv = 68.9%, p = 0.0002 and 0.0004, respectively) and 3 h p.i. (ctr: mv = 42.4%; OND: mv = 67.6%;

R252: mv = 65.4%, p = 0.0263 and 0.0066, respectively). In the CDV-infected monocytes also the CD14 expression intensities were 2.5-fold higher directly p.i.

(ctr: mv = 659; OND: mv = 1621; R252: mv = 1648, p = 0.0019 and 0.0004, respectively) and 4.8-fold higher 3 h p.i. (ctr: mv = 289; OND: mv = 1424;

R252: mv = 1379, p = 0.0008 and 0.0003, respectively) compared to the non-infected control (see Fig. 1).

Detection and virus load of CDV in canine monocytes

Monocytes could be experimentally infected with both CDV strains. OND virus could be detected in 33% of the monocytes directly p.i. and 23% 3 h p.i. whereas R252 was demonstrated in 61% directly p.i. and 50% 3 h p.i. Therefore, the virulent R252 strain infected significantly more monocytes than the attenuated OND (p = 0.0475; see Fig. 2).

The virus load of the R252-infected monocytes directly p.i. was 2.3fold higher than the one of the OND-infected monocytes (R252 mfi: mv = 108.2;

OND mfi: mv = 247.7, p = 0.0108) and 2.9fold higher 3 h p.i. (R252 mfi: mv = 88.9;

OND mfi: mv = 259.2) (see Fig. 2).

ROS-production in CDV infected monocytes

Without triggering only a low percentage of monocytes produced ROS directly p.i.

(ctr: mv = 5.6%; OND: mv = 4.0%; R252: mv = 3.7%) whereas a much higher percentage of the monocytes produced ROS after triggering with PMA (ctr: mv = 37.2%; OND: mv = 40.0%; R252: mv = 41.4%). Directly p.i. the ROS-generation was significantly higher in the OND-infected monocytes in comparison to the ctr. (p = 0.0109). At 3 h p.i., monocytic ROS generation was approximately 42% (mv of all groups) without triggering (ctr: mv = 35.4%;

OND: mv = 45.0%; R252: mv = 46.4%) and was not further enhanced by triggering with PMA (ctr: mv = 44.2%; OND: mv = 43.6%; R252: mv = 41.6%).

The ROS generation intensity was low in the non-triggered monocytes directly p.i.

(ctr mfi: mv = 198; OND mfi: mv = 163; R252 mfi: mv = 154) and enhanced after triggering (ctr mfi: mv = 781; OND mfi: mv = 514; R252 mfi: mv = 474). Three hours p.i. the ROS generation intensity was even lower in the non-triggered monocytes (ctr mfi: mv = 131; OND mfi: mv = 97; R252 mfi: mv = 107) compared to directly p.i.

but higher in the triggered monocytes (ctr mfi: mv = 1252; OND mfi: mv = 933;

R252 mfi: mv = 428) compared to directly p.i. (see Fig. 3). The ROS generation intensity is significantly higher in the control compared to the R252-infected triggered monocytes directly p.i. and 3 h p.i. (p = 0.0463 and p = 0.0431, respectively).

In OND-infected monocytes the up-regulated expression of CD14 correlated with the up-regulated percentage of ROS-producing monocytes after PMA triggering directly p.i. (p = 0.036). The increased virus load of R252-infected monocytes directly p.i.

correlated with the elevated percentage of ROS-generating non-triggered monocytes (p = 0.0366).

DISCUSSION

Acute canine distemper virus infection leads to immunosuppression and demyelination in the CNS (Vandevelde et al., 1982). In this process microglia cells are activated and act as antigen presenting cells through their MHC-molecules and their surface molecule CD1c. They futhermore stimulate T-cell activation and enhance their ROS generation (Stein et al., 2004a). Microglial generation of ROS could play an important role in the pathogenesis of demyelination (Stein et al., 2004b). During the course of the CDV infection peripheral blood monocytes were recruited to invade the CNS to support microglia cells in their defence against invaders (Griot-Wenk et al., 1991a, b; Wisniewski et al., 1972).

Following their crossing of the blood-brain-barrier monocytes transform into macrophages which makes them hardly distinguishable from microglia since their immunophenotype adapts to microglia cells. However, not much is known about ROS generation of CDV-infected peripheral blood monocytes. Therefore, the aim of this study was to prove the hypothesis that peripheral blood monocytes have the capacity to generate ROS and whether different CDV strains may influence the extent of the ROS generation.

The isolated monocytes were identified in flow cytometry by their morphology concerning size and complexity and their expression of CD14. They were infected by two different CDV strains: the attenuated OND which is known not to cause any neurological lesions in the CNS (Stettler et al., 1997) and the R252 which is known to cause demyelinating lesions in the CNS (Summers et al., 1984). An infection of the monocytes with either OND or R252 induced an up-regulation of the CD14 expression as well as an enhanced CD14 expression intensity. The CD14 expression intensity was higher 3 h p.i. compared to directly p.i. Although another CDV strain was used (A75/17), an up-regulation of CD14 expression on monocytes

was also found in a longitudinal study with in vivo CDV infected dogs (Stein et al., 2008). The up-regulation of the percentage of CD14⁺ monocytes was noted in the second week p.i. and remained constant until the end of the experiment (5 weeks p.i.). Therefore, in that study differences in CD14 expression were not evaluated within the first hours post CDV infection. Interestingly, the group of dogs with no or only mild clinical signs and no CNS lesions showed 74% CD14⁺ monocytes whereas in the group of dogs with severe clinical signs and demyelinating lesions in the CNS only 54% were CD14⁺.

Both virus strains were able to infect the isolated monocytes but the percentage of infected monocytes and virus load of the R252-infected monocytes was twice as high as the OND-infected monocytes p.i. and 3 h p.i.. The infection of the two different strains modulates the CD14 expression and the ROS generation. Initiation of the CD14/TLR4 dependend pathway is necessary for activating the innate immunity.

The results of the study show that ROS is generated by CDV-infected peripheral blood monocytes but depending on the virus strain the extent of ROS generation is different. Whereas infection with the vaccine strain OND results in a generous ROS generation, the ROS generation of the R252-infected monocytes seems to be restricted. ROS can cause oxidative damage to proteins, lipids and nucleic acids (Gilgun-Sherki et al., 2004) which might result in neurodegeneration (Lu et al., 2000). Therefore, also small amounts of ROS have to be seen critical in the vulnerable environment of the CNS. Interestingly, the demyelinating strain R252 just mildly triggers the monocytes to generate ROS which results in a higher chance to escape the immune system and enter the CNS but once there the small amount of ROS might be enough to cause damage to the oligodendrocytes because of their specific vulnerability for ROS (Griot et al., 1990).

Another interesting finding is that the percentage and the virus load of R252-infected monocytes in comparison to the OND-infected monocytes is twice as high but the ROS production is decreased in the R252-infected monocytes. Decreased ROS generation will lead to reduced virus clearance and enable the possibility of escaping the immune system to cause virus persistence.

This feature supports findings of other studies that virulent CDV strains show the ability to cause viral persistence in the CNS (Zurbriggen et al., 1995a; Zurbriggen et al., 1995b).

The generous ROS generation of the vaccine strain OND activates the immune system so antibodies will be produced to eliminate CDV infection in the ongoing life of a dog.

Monocytes can activate the innate immunity through the CD14/TLR4-dependent pathway to fight viral infections (Rolland et al., 2006). Although this mechanism is aimed at elimination of the virus, the activation can result in triggering neurodegeneration (Lehnardt et al., 2003). It has been reported that patients suffering from multiple sclerosis, which is a demyelinating neurodegenerative disease in humans, show an increase of CD14 to trigger the innate immunity (Brettschneider et al., 2002).

Since monocytes are recruited to migrate into the CNS in case of a CDV infection to explore there potential to generate ROS, they could play a crucial part in the pathogenesis of demyelination in the CNS, probably even to a higher degree than microglia.

Conclusions

This study confirms that CDV infection triggers peripheral blood monocytes to generate ROS, so once recruited to the CNS there ROS generation may play a pivotal role in the pathogenesis of demyelination in acute canine distemper virus infection.

Otherwise, the extent of ROS generation depends on the virus strain and interestingly, the virulent strain R252 triggers less ROS generation than the attenuated OND strain and is below the ROS generation potential of non-infected monocytes. The suppression of monocytic ROS generation could be a mechanism

Im Dokument Untersuchungen über den Einfluss der akuten Staupevirusinfektion auf die Funktion ex vivo kultivierter kaniner Monozyten (Seite 62-82)