Streszczenie

Wstęp: Przedmiotem pracy było określenie syntezy IFN-γ, IL-2, TNF-α (Th1/Tc1) oraz IL-4 (Th2/Tc2) na poziomie pojedynczych limfocytów T CD4⁺ i T CD8⁺ w przebiegu zakażenia myszy BALB/c i C57BL/6 szczepem Moscow wirusa ektromelii (ECTV-MOS).
Materiał/Metody: Syntezę IFN-γ, IL-2, TNF-α oraz IL-4 przez limfocyty T CD4⁺ i T CD8⁺ w drenujących węzłach chłonnych (DLN) i śledzionach myszy BALB/c i C57BL/6 oceniano metodą barwienia wewnątrz­komórkowego z analizą cytometryczną.
Wyniki: Przedstawione wyniki wskazują na wzrost odsetka limfocytów T CD8⁺ syntetyzujących IFN-γ we wczesnej fazie zakażenia ECTV-MOS jedynie w DLN i śledzionie myszy C57BL/6. Ponadto synteza IL-2 przez limfocyty T CD4⁺ i T CD8⁺ wystąpiła u tych zwierząt wcześniej i była silniej­sza w porównaniu do myszy BALB/c. Wzrost syntezy TNF-α przez limfocyty T CD4⁺ i T CD8⁺ obserwowano zwłaszcza w DLN zakażonych zwierząt. Nie wykazano różnic w odsetku limfocy­tów T syntetyzujących IL-4 (Th2 i Tc2) u obu szczepów myszy podczas zakażenia ECTV-MOS.
Wnioski: Przedstawione wyniki potwierdziły, że myszy C57BL/6 rozwijają silną odpowiedź Th1 oraz Tc1 we wczesnej fazie zakażenia ECTV-MOS. Myszy BALB/c, które przeżyły ostrą postać choroby, były również w stanie wykształcić dostatecznie silną odpowiedź komórkową przeciwko ECTV-MOS, jednak skuteczna eliminacja wirusa u wrażliwych szczepów myszy może następować wol­niej niż u myszy C57BL/6. Niewielka czułość metody wewnątrzkomórkowego wykrywania IL-4 za pomocą cytometrii przepływowej nie pozwoliła na ocenię różnic w syntezie IL-4 przez komór­ki Th2 i Tc2 między wrażliwymi a opornymi szczepami myszy podczas zakażenia ECTV-MOS.

Słowa kluczowe:wirus ektromelii • ospa myszy • cytokiny • limfocyty T

Summary

Background: The purpose of the study was to evaluate synthesis of IFN-γ, IL-2, TNF-α (Th1/Tc1) and IL-4 (Th2/Tc2) at CD4⁺ T and CD8⁺ T cell level in BALB/c and C57BL/6 mice in the course of infec­tion with ectromelia virus Moscow strain (ECTV-MOS).
Material/Methods: Synthesis of IFN-γ, IL-2, TNF-a and IL-4 in CD4⁺ T and CD8⁺ T cells in draining lymph nodes (DLNs) and spleens of BALB/c and C57BL/6 mice was detected by intracellular staining and flow cytometry analysis. r>Results: Our results showed an increase in percentage of IFN-γ-synthesizing CD8⁺ T cells only in DLNs and spleens of C57BL/6 mice at the early stages of infection. Moreover, synthesis of IL-2 by CD4⁺ and CD8⁺ T cells occurred earlier and was stronger in C57BL/6 mice compared to BALB/c mice. The increase in TNF-α synthesis by CD4⁺ T and CD8⁺ T cells was detected mainly in DLNs of infected animals. We did not observe any changes in the percentage of IL-4-synthesizing T cells (Th2 and Tc2) during ECTV-MOS infection in both strains of mice. r>Conclusions: Results presented in this study confirmed that during the early phase of infection, C57BL/6 mice mounted a strong Th1 and Tc1 immune response against ECTV-MOS. BALB/c mice that survi­ved the acute stage of mousepox, were able to mount an adequate cellular response to ECTV-MOS, however successful elimination of the virus in susceptible mice may occur more slowly compared to resistant strains of mice. Intracellular detection of IL-4 by flow cytometry was not sensitive enough to distinguish the differences in IL-4-synthesizing Th2 and Tc2 cells between susceptible and resistant strains of mice during ECTV-MOS infection.

Key words:ectromelia virus • mousepox • cytokines • T lymphocytes

Introduction

Activation of T cells results in production of cytokines that play a crucial role in orchestrating the immune response re­quired to control and clear invading pathogens. Differences in cytokine secretion profiles, expression of cell surface chemokine receptors and nuclear transcription factors are exhibited by various subpopulations of CD4⁺ T cells expo­sed to different environmental stimuli [7,16,17,27,31]. The type of T helper (Th) immune response determines the on­set of many viral infections. Early production of cytokines such as interferon (IFN)-α/β, IFN-γ, interleukin (IL)-2 and IL-12p40 induces a strong cellular immune response, that is critical for virus clearance and full recovery (profile as­sociated with Th1 cells). On the other hand, in many vi­ral infections generation of Th2 immune response, asso­ciated with IL-4, IL-5, IL-10 and IL-13 production, does not provide protection and often leads to a progressive and fatal disease [18,19].

Similar to CD4⁺ T cells, cytotoxic CD8⁺ T lymphocytes (CTLs) can be classified into at least two distinct subsets based on their cytokine-secreting profiles and chemokine receptors [22]. Type 1 CD8⁺ T cells (Tc1) predominantly secrete IFN-γ, IL-2 and tumor necrosis factor (TNF)-α, whereas Tc2 effector cells preferentially secrete IL-4, IL-5, IL-6 and IL-10 [1,22]. Tc1 cells have a capacity to migra­te to inflamed tissues and kill tumor or virus-infected cells [12]. Tc2 cells were shown to have a reduced antiviral ac­tivity in vivo not due to reduced secretion of IFN-γ, but due to alteration in homing to the target organ [30]. The presence of Tc2 cells often correlates with disease severi­ty and progression in chronic viral infections, cancer, and neurologic and autoimmune diseases [12]. The importance of Tc2 cells in progression of chronic pathologies may be explained by their capacity to modify the function of den­dritic cells (DCs) and favor differentiation of naive CD4⁺ and CD8⁺ T cells to type 2 cells [12].

Alteration of the Th1/Th2 and Tc1/Tc2 balance is obse­rved in many pathological conditions including immune­-mediated inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, progressive systemic sclerosis, sys­temic lupus erythematosus, and chronic obstructive pul­monary disease [15,29]. In many viral and bacterial infec­tions generation of inappropriate type of immune response can exacerbate disease and lead to inefficient elimination of the infectious agent.

Ectromelia virus (ECTV) is a member of the Poxviridae family, genus Orthopoxvirus and is a causative agent of mousepox, a disease called “smallpox of mice”. ECTV is closely related to variola virus (VARV) – a causative agent of smallpox responsible for millions of deaths in the histo­ry of mankind. Mousepox model is arguably the most su­itable small animal model for the study of orthopoxvirus infections including smallpox. Moreover, mousepox mo­del is used extensively to investigate the pathogenesis of generalized viral infections, genetic resistance to disease and viral immunology [2,3,4,13,20,25].

Among various inbred strains of mice there is genetically determined susceptibility to lethal infection with ECTV. Thus, BALB/c, DBA (both H-2^d haplotype) and C3H (H-2^k) mice have been classified as genetically susceptible, whi­le C57BL/6, C57BL/10 and 129/Sv (all H-2^b) mice are re­sistant to lethal infection with ECTV [2]. Strain variations in resistance to lethal form of mousepox are controlled by multiple, unlinked, dominant genes, including the natu­ral killer (NK) cell genes complex, the fifth component of complement (C5) gene, major histocompatibility complex (MHC) genes and selectin gene complex [5,6]. Resistant strains of mice recover from primary ECTV infection due to mobilization of multiple mechanisms of innate and ada­ptive immune response, including production of IFNs, acti­vity of NK cells and macrophages (Mo), activation of com­plement system, early and strong activation of virus-specific CTLs and production of antiviral antibodies (Abs) [3,13,20].

Chaudhri and colleagues [4] have shown that during ECTV infection, resistant mice produce high level of Th1 cytokines, such as IFN-γ, IL-2 and TNF-α, which was associated with strong cellular immune response and full recovery. On the contrary, in susceptible strains of mice the production of Th1 cytokines was absent or at a very low level, leading to weak and delayed cellular response resulting in high mortality ra­tes [4]. However, the production of Th1/Th2 and Tc1/Tc2 cytokines by CD4⁺ and CD8⁺ T cells, respectively, during mousepox has not been fully investigated. Thus, the purpose of the present study was to evaluate the synthesis of IFN-γ, IL-2, TNF-α (Th1/Tc1) and IL-4 (Th2/Tc2) by CD4⁺ and CD8⁺ T cells in BALB/c and C57BL/6 mice during prima­ry infection with Moscow strain of ECTV (ECTV-MOS).

Materials and Methods

Animals

Inbred, male BALB/c (H-2^d) and C57BL/6 (H-2^b) mice, be­tween 8-12 weeks of age, were purchased from the Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw, Poland. Animals were acclimated for 1 week before experiments and were allowed ad libitum access to food and water. Mice used in studies described in this report were sacrificed by cervical dislocation. All expe­riments were conducted under the permission granted by the 3^rd Local Ethical Commission for Animal Research at the Warsaw University of Life Sciences-SGGW (No. 61/2009).

Virus

Highly infectious ECTV-MOS (ATCC 1374) was grown in Vero cells (ATCC CCL-81) monolayer, then titrated and stored in aliquots at -80°C until used. Fifty plaque forming units (PFU) of ECTV-MOS were injected into the hind foot pad (f.p.) in a total volume of 20 µl/f.p of an anesthetized mouse. BALB/c and C57BL/6 mice injected with sterile phosphate-buffered saline (PBS) served as ne­gative controls.

Antibodies

The following monoclonal antibodies (mAbs) directed aga­inst mouse leukocyte surface markers and mouse intracel­lular cytokines in appropriate combinations were used: an­ti-CD4-PerCP (H129.9), anti-CD8-PE or anti-CD8-FITC (53-6.7), anti-IFN-γ-PE (XMG1.2), anti-IL-2-PE (JES6-5H4), anti-TNF-α-FITC (MP6-XT22) and anti-IL-4-FITC (11B11). The antibodies were initially titrated to obtain the optimal concentration. All antibodies and appropria­te isotype controls were purchased from BD Biosciences.

Reagents

Phorbol 12-myristate 13-acetate (PMA) (Sigma) and io­nomycin (Sigma) were used for nonspecific stimulation of cytokine production. GolgiPlug reagent (containing brefeldin A that has an inhibitory effect on protein secre­tion) and reagents for cell fixation and permeabilization (Cytofix/Cytoperm and Perm/Wash) were obtained from BD Biosciences.

Preparation of single cell suspension

Draining lymph nodes (DLNs – popliteal and superficial inguinal) and spleens were aseptically removed at 7, 14 and 21 day post infection (d.p.i.) or from uninfected control animals. The number of animals per experimental group was as follows: control BALB/c mice (n=5 to 8); control C57BL/6 mice (n=5 to 9); BALB/c mice at 7 d.p.i. (n=6 to 11); C57BL/6 mice at 7 d.p.i. (n=5 to 6); BALB/c mice at 14 d.p.i. (n=5 to 6); C57BL/6 mice at 14 d.p.i. (n=5 to 11); BALB/c mice at 21 d.p.i. (n=5 to 6); C57BL/6 mice at 21 d.p.i. (n=5 to 10). The isolated organs were mechanical­ly disaggregated by passing through stainless steel sieves. After erythrocyte depletion the viability of cells was as­sessed using 0.4% trypan blue exclusion test.

Enumeration of cytokine-synthesizing cells by intracellular staining

Freshly isolated cells were restimulated in vitro with iono­mycin/PMA in accordance with BD Biosciences protocols and literature data [8,9]. Briefly, cells were placed in 96-well plates and stimulated using PMA (50 ng/ml) and io­nomycin (500 ng/ml) in the presence of brefeldin A (10 µg/ml) for 4 hrs at 37oC in 5%CO₂. Later, cells were washed in PBS supplemented with 3% fetal bovine serum (FBS) and blocked for nonspecific binding in 30% FBS for 30 min on ice. Surface staining was then performed using mAbs anti-CD4 and anti-CD8 followed by intracellular staining with Cytofix/Cytoperm kit in accordance with the manu­facturer’s instructions. Briefly, cells were fixed and perme­abilized with Cytofix/Cytoperm solution for 25 min on ice followed by washing in Perm/Wash solution. Next, cells were stained for 30 min on ice with mAbs against intra­cellular cytokines: IFN-γ, IL-2, TNF-α or IL-4 and finally washed in Perm/Wash solution and 3% FBS/PBS, fixed in 2% paraformaldehyde (PFA) in PBS and analyzed within 24 hrs by flow cytometry.

Flow cytometry acquisition and analysis

Percentage of cytokine-synthesizing cells was measured by FACSCalibur flow cytometer (Becton Dickinson) and ana­lyzed by CellQuest software. Data from 2×10⁴ and 5×10⁴ events were acquired for each sample. Nonlymphoid and dead cells were excluded by appropriate gating based on forward (FSC) and side (SSC) scatter profiles. Single-cell cytokine synthesis was assessed in a gate set on lympho­cyte population using FSC and SSC characteristics.

Statistical analysis

Percentage of cytokine-synthesizing CD4⁺ and CD8⁺ T cells was given as mean ±SD (standard deviation). Differences between uninfected control mice and infected animals at indicated time points were calculated using the Student’s t-test (in case of normal data distribution with equal varian­ce) or Mann-Whitney U-test (STATISTICA 6.0 software, StatSoft). Statistical significance was determined at P<0.05.

Results

We examined the synthesis of IFN-γ, IL-2, TNF-α and IL-4 by CD4⁺ and CD8⁺ T cells in both susceptible (BALB/c) and resistant (C57BL/6) inbred strains of mice during ECTV-MOS infection. 4 hrs were chosen as an optimal time to achieve synthesis of cytokines in in vitro restimulation of isolated cells with ionomycin and PMA (data not shown). Unstimulated cells incubated in vitro for 4 hrs in the pre­sence of transport inhibitor (brefeldin A) served as a con­trol of spontaneous cytokine synthesis (Figure 1).

Figure 1. Representative dot plots show spontaneous synthesis of IFN-γ (A) by CD8⁺ T cells and IL-2 (B), TNF-α (C) and IL-4 (D) by CD4⁺ T cells isolated from spleens of uninfected BALB/c (D) or C57BL/6 (A, B, C) mice. Cells were incubated for 4 hrs in the presence of brefeldin A. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocyte population defined by FSC and SSC parameters

Synthesis of IFN-γ by CD4⁺ and CD8⁺ T cells in C57BL/6 mice during ECTV-MOS infection

Intracellular detection of IFN-γ in T lymphocytes of BALB/c mice in the early stages of mousepox (until 14 d.p.i.) has been previously described [25]. For that reason only results concerning the synthesis of IFN-γ by T cells in DLNs and spleens of C57BL/6 mice infected with ECTV-MOS are reported here.

During ECTV-MOS infection of C57BL/6 mice, higher percentage of CD8⁺IFN-γ⁺ T cells than CD4⁺IFN-γ⁺ T cells was observed (Figure 2). In DLNs and spleens of C57BL/6 mice at 7 d.p.i. a statistically significant (P<0.05) incre­ase in percentage of CD8⁺ T cells synthesizing IFN-γ was observed compared to uninfected control animals (18% vs 14% and 30% vs 17%, respectively) (Figure 2B).

Figure 2. Percentage of IFN-γ-synthesizing CD4⁺ (A) and CD8⁺ (B) T cells in DLNs and spleens of C57BL/6 mice during mousepox. (C) Representative dot plots show synthesis of IFN-γ by CD8⁺ T cells in spleens of C57BL/6 mice during ECTV-MOS infection. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocytes (* P=<0.05, ** P=<0.01)

The strongest synthesis of IFN-γ by CD4⁺ and CD8⁺ T cells was detected at 14 d.p.i. in both tissues. Moreover, at 14 d.p.i. there was a significantly higher (P<0.05) percen­tage of CD4⁺IFN-γ⁺ T cells in DLNs and CD8⁺IFN-γ⁺ T cells in DLNs and spleens of C57BL/6 mice compared to BALB/c mice [25]. The percentage of CD8⁺ T cells syn­thesizing IFN-γ in DLNs and spleens of C57BL/6 mice started to decrease during recovery, whereas in BALB/c mice it remained high until day 21 p.i. (data not shown).

Differences in percentage of IL-2-synthesizing T cells between BALB/c and C57BL/6 mice during ECTV-MOS infection

During early stages of mousepox (7 d.p.i.) the percentage of IL-2-synthesizing CD4⁺ T cells in DLNs and spleens of BALB/c mice was lower and their appearance was delayed compared to the C57BL/6 group (Figure 3A, 3C). Increase in the percentage of IL-2-synthesizing CD4⁺ T cells in DLNs and spleens of both strains of mice peaked on day 14 p.i. and remained high until day 21 in spleens of infected ani­mals. Meanwhile, the percentage of IL-2-synthesizing CD8⁺ T cells was approximately 2-fold lower than that of CD4⁺ T cells during ECTV-MOS infection (Figure 3B). The percen­tage of IL-2-synthesizing CD8⁺ T cells varied between the two inbred strains of mice. In DLNs and spleens of BALB/c mice the highest percentage of CD8⁺IL-2⁺ T cells was ob­served at 14 d.p.i. and remained high until 21 d.p.i., where­as in C57BL/6 a statistically significant (P<0.05) increase in percentage of IL-2-synthesizing CD8⁺ T cells was obse­rved as early as day 7 p.i. and thereafter decreased gradual­ly. In spleens of BALB/c mice at day 14 and 21 p.i., higher percentage of IL-2⁺CD8⁺ T cells was observed compared to C57BL/6 mice (Figure 3). High synthesis of this Tc1 cytoki­ne in BALB/c mice continued until day 21 p.i. (Figure 3B).

Figure 3. Percentage of IL-2-synthesizing CD4⁺ (A) and CD8⁺ (B) T cells in DLNs and spleens of BALB/c and C57BL/6 mice during mousepox. (C) Representative dot plots show synthesis of IL-2 by CD4⁺ T cells in spleens of BALB/c and C57BL/6 mice during ECTV-MOS infection. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocytes (* P=<0.05, ** P=<0.01)

BALB/c and C57BL/6 mice displayed similar percentage of TNF-α-synthesizing T cells during ECTV infection

In DLNs and spleens of BALB/c and C57BL/6 mice we found similar percentage of TNF-α-synthesizing T cells throughout the course of infection (Figure 4). The percen­tage of TNF-α-synthesizing CD4⁺ and CD8⁺ T cells rema­ined unchanged until 7 d.p.i. in both tissues in BALB/c and C57BL/6 mice. In DLNs of BALB/c and C57BL/6 mice at 14 d.p.i. a statistically significant (P<0.01) increase in TNF-a-synthesizing CD4⁺ T cells was observed compared to uninfected animals (67% vs 38% and 60% vs 32%, respec­tively) (Figure 4A). In spleens, at this time point of infec­tion a significant (P<0.05) increase in TNF-α-synthesizing CD4⁺ T cells was also detected. However the increases were smaller than those found in DLNs (43% vs 32% in BALB/c and 54% vs 40% in C57BL/6 mice, respectively). The per­centage of CD4⁺ T cells synthesizing TNF-α in DLNs and spleens of BALB/c and C57BL/6 mice remained high until 21 d.p.i. In both organs of the two strains of mice a statisti­cally significant (P<0.01) increase in TNF-α-synthesizing CD8⁺ T cells was observed at 14 d.p.i. compared to unin­fected animals (Figure 4B). In DLNs the percentage of TNF-α⁺CD8⁺ T cells declined by day 21 p.i., whereas in spleens of BALB/c mice it peaked on day 21 p.i.

Figure 4. Percentage of TNF-α-synthesizing CD4⁺ (A) and CD8⁺ (B) T cells in DLNs and spleens of BALB/c and C57BL/6 mice during mousepox. (C) Representative dot plots show synthesis of TNF-α by CD4⁺ T cells in spleens of BALB/c and C57BL/6 mice during ECTV-MOS infection. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocytes (* P=<0.05, ** P=<0.01)

Flow cytometric analysis did not reveal changes in intracellular IL-4 production by T cells during mousepox

Although CD4⁺ T cells were the main subpopulation of T cells synthesizing IL-4, during ECTV-MOS infection of BALB/c and C57BL/6 mice no statistically significant changes in the percentage of IL-4-synthesizing CD4⁺ and CD8⁺ T cells were detected in DLNs and spleens compared to uninfected group of animals (Figure 5). Moreover, in both T cell subsets no statistically significant changes in mean fluorescence inten­sity (MFI) of IL-4 expression were observed between control and infected BALB/c and C57BL/6 mice (data not shown).

Figure 5. Percentage of IL-4-synthesizing CD4⁺ (A) and CD8⁺ (B) T cells in DLNs and spleens of BALB/c and C57BL/6 mice during mousepox. (C) Representative dot plots show synthesis of TNF-α by CD4⁺ T cells in spleens of BALB/c and C57BL/6 mice during ECTV-MOS infection. Numbers within quadrants represent the percentage of positive cells for a given marker within the gate for lymphocytes (* P=<0.05, **P=<0.01)

Discussion

Cytokine production by the responding lymphocytes di­rects the development of antigen-specific immune response against the infectious agent. The secretion of various cy­tokines may result in protective cellular immune response but on the other hand may facilitate virus dissemination by suppression of the mechanisms intended to eliminate the infectious agent. In the present study we evaluated the synthesis of Th1/Th2 and Tc1/Tc2 cytokines by CD4⁺ and CD8⁺ T cells, respectively, in both susceptible (BALB/c) and resistant (C57BL/6) strains of mice infected with hi­ghly virulent ECTV-MOS. For single-cell level evaluation of cytokine synthesis we used intracellular staining tech­nique followed by flow cytometric analysis.

Our results indicated that there are distinct differences in the percentage of Th1/Tc1 cytokine-synthesizing cells in resi­stant and susceptible mice during mousepox (Figures 2,3,4). In C57BL/6 mice the synthesis of Th1/Tc1 cytokines (IFN-g and IL-2) occurred earlier than in BALB/c mice [25]. This may reflect strong proliferation of CD4⁺ and CD8⁺ T cells following antigen specific recognition in C57BL/6 mice, and in consequence, T cells migrate to effector sites such as the spleen, liver and other organs for virus elimination [4]. Moreover, during secondary infection with ECTV, DLNs are the sites where lymphocytes become activated and prolifera­te, and where infected cells are eliminated by memory CD8⁺ T cells acting as gatekeepers that directly kill targets infec­ted with ECTV [32]. Our results confirmed that anti-ECTV­-MOS defense mechanisms at the early stages of infection come into effect much earlier in resistant strains of mice such as C57BL/6 whereas BALB/c mice display a delay in genera­tion of the optimal antiviral cellular immune response [18,23].

Besides the differences between strains of mice concerning the synthesis of Th1/Tc1 cytokines in the initial phase of in­fection, the kinetics of its synthesis at later stages of infec­tion showed certain similarities (Figures 2,3,4). In both strains of mice the greatest synthesis of IFN-γ, IL-2 and TNF-α by CD4⁺ and CD8⁺ T cells in DLNs and spleens was observed at 14 d.p.i., when the clinical signs of mousepox were severe. IFN-γ was synthesized mainly by CD8⁺ T cells, IL-2 mainly by CD4⁺ T cells, whereas TNF-α was synthesized by both subsets of T cells at comparable levels. Our data on cytoki­ne synthesis support the notion that ECTV-MOS-specific CD8⁺ and CD4⁺ CTLs form the basis of cellular immune response at 14 d.p.i. in infected BALB/c [23] and C57BL/6 mice. However, susceptible BALB/c mice generated weaker Th1/Tc1 immune response than resistant C57BL/6 mice, which indeed led to recovery from mousepox, but could not be sufficient for complete elimination of viral particles in the infected host. It has been shown that ECTV causes a persi­stent infection in splenic DCs and Mo of BALB/c mice fol­lowing the acute infection [24]. Meantime IFN-γ is the ma­jor cytokine that activates Mo, increasing their activity to kill intracellular pathogens and viruses. Moreover, the acti­vity of IFN-γ can be inhibited at the level of receptor enga­gement by soluble IFN-γR homolog (vIFN-γR), a virocep­tor encoded by ECTV, which represents a strategy to evade the antiviral effects of this cytokine [6]. High production of Th1/Tc1 cytokine until 21 d.p.i. in BALB/c mice sugge­sts that in susceptible strains of mice the successful elimi­nation of the virus from the infected host through adaptive immune response occurs more slowly than in resistant mice.

IFN-γ is a critical effector cytokine responsible for ECTV elimination from all major organs, except the primary sites of infection, where viral clearance appears to be delayed [14]. Moreover, IFN-_γ produced by the CD4⁺ helper T cells is involved in the generation of optimal CD8⁺ CTLs response [18]. The importance of IFN-γ in the development of Th1 cytokine immune response and elimination of the virus has been shown using neutralizing mAbs. Treatment of C57BL/6 mice with neutralizing mAbs against IFN-γ resulted in enhanced spread to and efficient ECTV repli­cation in the spleen, lungs, ovaries and liver, transforming infection from a self-limiting disease to fulminant mouse­pox [4,14].

It has been reported that neutralizing TNF-α or IL-2 func­tion in C57BL/6 mice does not significantly affect viral re­plication and does not reverse the resistance to mousepox [4]. However, normal or immunodeficient mice infected with vaccinia virus (VACV) expressing IFN-γ or TNF-α re­vealed attenuation of virus-induced pathogenicity and rapid virus clearance [11]. It has been shown, that the antiviral ef­fect of TNF-α on ECTV depends on p55 and p75 TNF re­ceptors. Mice lacking p75 were significantly more suscepti­ble to ECTV and most mice succumbed to lethal infection [21]. TNF-α synergizes with IFN-γ to induce multiple anti­viral activities. Intriguingly, TNF-α production is cell-type restricted and cannot be substituted by other cellular sources. TNF-α produced by antigen-specific CD4⁺ and CD8⁺ T cells is involved in direct killing of infected cells and activation of inflammatory response [10]. In our studies, higher synthesis of TNF-α by T cells in DLNs of infected animals confirms that TNF-α secretion is organ specific and may represent uni­que requirements for distinct cytokines in T cell priming [4].

Intracellular detection of IL-4 did not reveal any statistically significant changes in the percentage of IL-4-synthesizing CD4⁺ (Th2) and CD8⁺ (Tc2) T cells during mousepox in both strains of mice (Figure 5). It is not excluded that flow cytometric analysis of intracellular IL-4 is not sufficient for this purpose due to its low sensitivity [28]. Meanwhile, it has been shown that susceptible strains of mice genera­te a Th2 cytokine response with IL-4 production [4], ho­wever the cellular origin of IL-4 has not been defined. It is known that IL-4 down-regulate the production of Th1 cytokines. This may explain why generation of IL-2 and IFN-γ is delayed at the early stages of mousepox in BALB/c mice compared to C57BL/6 mice. On the other hand it was shown, that the absence of IL-4 did not reverse the suscepti­bility of BALB/c mice to ECTV infection [4]. The immune responses to ECTV by both IL-4 gene knockout mice on a BALB/c background and BALB/c mice treated with neutra­lizing mAbs against IL-4 were similar to wild type BALB/c mice [4]. Moreover, inactivation of signal transducer and activator of transcription 6 (STAT6), which is responsible for IL-4-mediated differentiation events, was not sufficient for BALB/c mice to overcome ECTV infection. However, STAT6^-/- mice infected with ECTV displayed minimal le­sions in the vital organs and delayed mortality, what correla­ted with suppressed Th2 immunity [18]. Chaudhri and col­leagues [4] have shown that despite the differences in IL-4, IL-2, TNF-α and IFN-γ production in DLNs and spleens between different strains of mice, the expression of mRNA for those cytokines was comparable between BALB/c and C57BL/6 mice suggesting the existence of posttranscriptio­nal regulation of cytokine expression in vivo.

Recently, we have found that during early stages of mouse­pox in spleens of BALB/c mice there was an increase in IL-17A-producing CD4⁺ and CD8⁺ T cells [26]. Therefore, IL-17A may play a role in both elimination of viral partic­les or promoting the tissue damage during the acute phase of infection with ECTV-MOS [26].

In conclusion, our results confirmed that at early stages of ECTV-MOS infection, C57BL/6 mice mounted earlier and stronger type 1 cytokine response. When infected with small dose of virus (50 PFU) BALB/c mice were able to survive an acute phase of infection and were able to mount relatively strong and effective Th1/Tc1 immune response. In most cases, however, anti-ECTV-MOS defense mechanisms at the early stages of infection came into effect too late and for those re­asons susceptible strains of mice often succumbed to a disease.

REFERENCES

[1] Austin L.M., Ozawa M., Kikuchi T., Walters I.B., Krueger J.G.: The majority of epidermal T cells in Psoriasis vulgaris lesions can produce type 1 cytokines, interferon-gamma, interleukin-2, and tumor necrosis factor-alpha, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias is also measured in circulating blood T cells in psoriatic patients. J. Invest. Dermatol., 1999; 113: 752-759
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[2] Bhatt P.N., Jacoby R.O.: Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. I. Clinical responses. Lab. Anim. Sci., 1987; 37: 11-15
[PubMed]  

[3] Chaudhri G., Panchanathan V., Bluethmann H., Karupiah G.: Obligatory requirement for antibody in recovery from a primary poxvirus infection. J. Virol., 2006; 80: 6339-6344
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[4] Chaudhri G., Panchanathan V., Buller R.M.L., van den Eertwegh A.J.M., Claassen E., Zhou J., de Chazal R., Laman J.D., Karupiah G.: Polarized type 1 cytokine response and cell-mediated immunity determine genetic resistance to mousepox. Proc. Natl. Acad. Sci. USA, 2004; 101: 9057-9062
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[5] Delano M.L., Brownstein D.G.: Innate resistance to lethal mousepox is genetically linked to the NK gene complex on chromosome 6 and correlates with early restriction of virus replication by cells with an NK phenotype. J. Virol., 1995; 69: 5875-5877
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[6] Esteban D.J., Buller R.M.L.: Ectromelia virus: the causative agent of mousepox. J. Gen. Virol., 2005; 86: 2645-2659
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[7] Fazilleau N., Mark L., McHeyzer-Williams L.J., McHeyzer-Williams M.G.: Follicular helper T cells: lineage and location. Immunity, 2009; 30: 324-335
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[8] Fujita H., Nograles K.E., Kikuchi T., Gonzalez J., Carucci J.A., Krueger J.G.: Human Langerhans cells induce distinct IL-22-producing CD4⁺ T cells lacking IL-17 production. Proc. Natl. Acad. Sci. USA, 2009; 106: 21795-21800
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[9] Gallego A., Vargas J.A., Castejón R., Citores M.J., Romero Y., Millán I., Durántez A.: Production of intracellular IL-2, TNF-α, and IFN-γ by T cells in B-CLL. Cytometry B Clin. Cytom., 2003; 56: 23-29
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[10] Grivennikov S.I., Tumanov A.V., Liepinsh D.J., Kruglov A.A., Marakusha B.I., Shakhov A.N., Murakami T., Drutskaya L.N., Förster I., Clausen B.E., Tessarollo L., Ryffel B., Kuprash D.V., Nedospasov S.A.: Distinct and nonredundant in vivo functions of TNF produced by T cells and macrophages/neutrophils: protective and deleterious effects. Immunity, 2005; 22: 93-104
[PubMed]  [Full Text PDF]  

[11] Guidotti L.G., Chisari F.V.: Cytokine-mediated control of viral infections. Virology, 2000; 273: 221-227
[PubMed]  

[12] Iezzi G., Boni A., Deql’Innocenti E., Grioni M., Bertilaccio M.T., Bellone M.: Type 2 cytotoxic T lymphocytes modulate the activity of dendritic cells toward type 2 immune responses. J. Immunol., 2006; 177: 2131-2137
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[13] Karupiah G., Buller R.M.L., Van Rooijen N., Duarte C.J., Chen J.H.: Different roles for CD4⁺ and CD8⁺ T lymphocytes and macrophage subsets in the control of a generalized virus infection. J. Virol., 1996; 70: 8301-8309
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[14] Karupiah G., Fredrickson T.N., Holmes K.L., Khairallah L.H., Buller R.M.: Importance of interferons in recovery from mousepox. J. Virol., 1993; 67: 4214-4226
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[15] Kidd P.: Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern. Med. Rev., 2003; 8: 223-246
[PubMed]  [Full Text PDF]  

[16] King C., Tangye S.G., Mackay C.R.: T follicular helper (T_FH) cells in normal and dysregulated immune responses. Annu. Rev. Immunol., 2008; 26: 741-766
[PubMed]  

[17] Louten J., Boniface K., de Waal Malefyt R.: Development and function of T_H17 cells in health and disease. J. Allergy Clin. Immunol., 2009; 123: 1004-1011
[PubMed]  

[18] Mahalingam S., Karupiah G., Takeda K., Akira S., Matthaei K.I., Foster P.S.: Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus. Proc. Natl. Acad. Sci. USA, 2001; 98: 6812-6817
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[19] Mosmann T.R., Sad S.: The expanding universe of T-cell subsets: T_h1, T_h2 and more. Immunol. Today, 1996; 17: 138-146
[PubMed]  

[20] Moulton E.A., Atkinson J.P., Buller R.M.: Surviving mousepox infection requires the complement system. PLOS Pathog., 2008; 4: e1000249
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[21] Ruby J., Bluethmann H., Peschon J.J.: Antiviral activity of tumor necrosis factor (TNF) is mediated via p55 and p75 TNF receptors. J. Exp. Med., 1997; 186: 1591-1596
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[22] Sad S., Marcotte R., Mosmann T.R.: Cytokine-induced differentiation of precursor mouse CD8⁺ T cells into cytotoxic CD8⁺ T cells secreting Th1 or Th2 cytokines. Immunity, 1995; 2: 271-279
[PubMed]  [Full Text PDF]  

[23] Spohr Cespedes I., Toka F.N., Schollenberger A., Gieryńska M., Niemiałtowski M.: Pathogenesis of mousepox in H-2^d mice: evidence for MHC class I-restricted CD8⁺ and MHC class II-restricted CD4⁺ CTL antiviral activity in the lymph nodes, spleen and skin, but not in the conjunctivae. Microbes Infect., 2001; 3: 1063-1072
[PubMed]  

[24] Spohr de Faundez I., Gieryńska M., Niemiałtowski M.G., Malicka E., Popis A.: Ectromelia virus establishes a persistent infection in spleen dendritic cells and macrophages of BALB/c mice following the acute disease. Adv. Exp. Med. Biol., 1995; 378: 257-261
[PubMed]  

[25] Szulc L., Boratyńska A., Martyniszyn L., Niemiałtowski M.G.: Antigen presenting and effector cell cluster formation in BALB/c mice during mousepox: model studies. J. Appl. Microbiol., 2010; 109: 1817-1828
[PubMed]  

[26] Szulc L., Gieryńska M., Boratyńska A., Martyniszyn L., Niemiałtowski M.: Flow cytometric analysis of intracellular IL-17A in T-cells during ectromelia virus infection. Acta Virol., 2011; 55: 367-369
[PubMed]  

[27] Świst K., Pajtasz-Piasecka E.: The influence of transcription factors on CD4⁺ T cell differentiation. Postępy Hig. Med. Dośw., 2011; 65: 414-426
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[28] Tayebi H., Lienard A., Billot M., Tiberghien P., Hervé P., Robinet E.: Detection of intracellular cytokines in citrated whole blood or marrow samples by flow cytometry. J. Immunol. Methods, 1999; 229: 121-130
[PubMed]  

[29] Tzanakis N., Chrysofakis G., Tsoumakidou M., Kyriakou D., Tsiligianni J., Bouros D., Siafakas N.M.: Induced sputum CD8⁺ T-lymphocyte subpopulations in chronic obstructive pulmonary disease. Respir. Med., 2004; 98: 57-65
[PubMed]  

[30] Wirth S., van den Broek M., Frossard C.P., Hügin A.W., Leblond I., Pircher H., Hauser C.: CD8⁺ T cells secreting type 2 lymphokines are defective in protection against viral infection. Cell. Immunol., 2000; 202: 13-22
[PubMed]  

[31] Wojas J., Pajtasz-Piasecka E.: Dendritic cell – regulatory T-cell interaction. Postępy Hig. Med. Dośw., 2010; 64: 167-174
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[32] Xu R.H., Fang M., Klein-Szanto A., Sigal L.J.: Memory CD8⁺ T cells are gatekeepers of the lymph node draining the site of viral infection. Proc. Natl. Acad. Sci. USA, 2007; 104: 10992-10997
[PubMed]  [Full Text HTML]  [Full Text PDF]  

The authors have no potential conflicts of interest to declare.

Full text