Marek Niemiałtowski

Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies.

Ectromelia virus (EV), which causes mousepox, is a member of the orthopoxviruses that are defined as being able to suppress apoptosis. Caspase-3 is one of the key effector proteases which regulates the apoptotic cascade and which is responsible for DNA fragmentation observed during apoptosis. It is well known that viruses, especially poxviruses, can inhibit caspase activity. Here, we report that EV can regulate apoptosis in vitro, suppressing the activity of caspases recognizing the DEVD (Asp-Glu-Val-Asp) motif (caspase-3 and -7) before successful virus replication is completed. Caspase-3 activity measurement showed that an increase in caspase-3 activity preceded the peak of DNA fragmentation demonstrated by TUNEL staining of L929 and RK-13 cells. By using specific caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO and zVAD-fmk), we showed that caspase-3 and -7 (DEVDases) are major effector caspases during EV-induced apoptosis in permissive L929 and RK-13 cell cultures. Apoptosis in vivo seems to play an important role during viraemia as well as during the clearance of EV from genetically susceptible BALB/c (H-2(d)) mice. However, as shown by measurement of caspase-3 activity, caspase-3 protein detection and M30-antibody staining, both DEVDases seem to play an important role during EV clearance from draining lymph nodes and conjunctivae at 15 days p.i. up to 20 days p.i., whereas in the liver and spleen DNA fragmentation coexisted with viral multiplication and secondary viraemia. Apoptosis was DEVDase dependent only in the liver, while spleen DNA fragmentation observed between 5 and 10 days p.i. was caspase independent. Therefore, we conclude that DEVDase- (caspase-3- and caspase-7-) dependent apoptosis is an important mechanism regulating the resolution of EV infection.

Long‐lasting immunity by early infection of maternal‐antibody‐protected infants

Newborn higher vertebrates are largely immuno‐incompetent and generally survive infections – including poxviruses – by maternal antibody protection. Here, we show that mice survived epidemics as adults only if exposed to lethal orthopoxvirus infections during infancy under the umbrella of maternal protective antibodies. This implies that both the absence of exposure to infection during early infancy or of effective vaccination renders the population highly susceptible to new or old re‐emerging pathogens.

Beclin 1 is involved in regulation of apoptosis and autophagy during replication of ectromelia virus in permissive L929 cells.

Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.

Pathogenesis of mousepox in H-2d 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

Genetically sensitive mice (i.e. H-2(d) haplotype) infected with a natural mouse pathogen named ectromelia virus (EV) can develop a mousepox. Virus replicates well in the skin, next in the draining lymph nodes (DLNs) and then in the spleen and liver, where it may induce extensive necrosis with strong inflammatory reaction. It is well known from the studies defined on some other viruses that a correlation, functional link and powerful help exist between MHC class I-restricted CD8(+) and MHC class II-restricted CD4(+) virus-specific cytotoxic T lymphocytes (CTLs). However, in the case of mousepox the role of CD4(+) CTLs is still controversial and some reports support the notion that induction of EV-specific CD4(+) CTLs is nonessential for the generation of virus-specific immune response. Consequently, this study was designed to evaluate EV-specific CD8(+) and CD4(+) CTL activity in the DLNs, spleen, skin and conjunctivae of BALB/c (H-2(d)) mice at 7 and 14 days p.i. with Moscow strain of EV. By using bulk cytotoxicity assay and immunosurgery of effector T cells with mAb specific for CD4(+) and/or CD8(+) T cells our data show that EV-specific CD8(+) CTLs predominated in DLNs and spleen at 7 days (67 and 66% of total CTLs, respectively) and 14 days p.i. (63 and 69% of total CTLs, respectively). In contrast, we found that EV clearance from the cutaneous lesions during mousepox is CD4(+) CTL-dependent at 7 days p.i. (59% of total CTLs), whereas at 14 days p.i. CD8(+) CTLs predominated in the epidermis, accounting for 72% of the total EV-specific CTLs. Our studies showed that the population of EV-specific CTLs is heterogeneous and contains cells of both phenotypes: CD8(+) and CD4(+). However, these effector cells did not express a similar tendency in cytotoxic activity in the DLNs, spleen and skin in comparison to the conjunctivae where EV-specific CD8(+) and CD4(+) CTLs were not detected at 7 days p.i. and at peak of mousepox conjunctivitis (14 days p.i.). Our results are discussed in terms of the value of EV to study antiviral CTL responses in the genetically susceptible host.

Modulation of NF-кB transcription factor activation by Molluscum contagiosum virus proteins.

Molluscum contagiosum virus is a human and animal dermatotropic pathogen, which causes a severe disease in immunocompromised individuals. MCV belongs to the Poxviridae family whose members exert immunomodulatory effects on the host antiviral response. Poxviruses interfere with cell signaling pathways that lead to the activation of nuclear factor кB, a pleiotropic transcription factor which is crucial for regulation of the immune response, the cell cycle and apoptosis. In resting cells, NF-κB is present in the cytoplasm, where it is associated with inhibitor κB. Upon stimulation by activators, such as proinflammatory cytokines and bacterial or viral products, the inhibitory protein undergoes phosphorylation and proteasomal degradation. NF-κB, in turn, translocates to the nucleus, where it regulates the transcription of various genes that are essential for processes mentioned above. Since poxviruses replicate exclusively in the cell cytoplasm, NF-кB became a good target for poxviral immunomodulation. MCV encodes various proteins which interfere with the signaling pathways that lead to the activation of NF-κB. Ligand inhibitor encoded by MCV, MC54, binds interleukin-18 and inhibits interferon-γ production. Other MCV proteins, MC159 and MC160, belong to intracellular inhibitors of NF-κB and are members of viral FLICE-inhibitory proteins (vFLIPs). MC159 protein encoded by MCV was shown to inhibit apoptosis of virus-infected cells. Such interactions serve immune evasion and are responsible for the persistence of MCV.

Crosstalk between autophagy and apoptosis in RAW 264.7 macrophages infected with ectromelia orthopoxvirus.

Several studies have provided evidence that complex relationships between autophagic and apoptotic cell death pathways occur in cancer and virus-infected cells. Previously, we demonstrated that infection of macrophages with Moscow strain of ectromelia virus (ECTV-MOS) induces apoptosis under in vitro and in vivo conditions. Here, we found that autophagy was induced in RAW 264.7 cells during infection with ECTV-MOS. Silencing of beclin 1, an autophagy-related gene, reduced the percentage of late apoptotic cells in virus-infected RAW 264.7 macrophages. Pharmacological modulation of autophagy by wortmannin (inhibitor) or rapamycin (inductor) did not affect or cause increased apoptosis in ECTV-MOS-infected RAW 264.7 cells, respectively. Meantime, blocking apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, increased the formation of autophagosomes in infected macrophages. Taken together, three important points arise from our study. First, autophagy may co-occur with apoptosis in RAW 264.7 cells exposed to ECTV-MOS. Second, at later stages of infection, autophagy may partially participate in the execution of macrophage cell death by enhancing apoptosis. Third, when apoptosis is blocked infected macrophages undergo increased autophagy. Our results provide new information about the relationship between autophagy and apoptosis in ECTV-MOS-infected macrophages.

Ectromelia Virus Establishes a Persistent Infection in Spleen Dendritic Cells and Macrophages of BALB/c Mice following the Acute Disease

The EV initially replicates in the skin and regional lymph nodes of mice. Next, it reaches the spleen and liver what might lead to death of mice due to irreversible damage of these organs1,2. However, some mice can survive acute disease (at peak between 5 and 10 days p.i.), and harbour EV for several months. To understand how EV avoid immune surveillance and persist, we have studied spleen dendritic cells (DC), macrophages (M0), T and B lymphocytes of the BALB/c mice up to 60 days p.i. The number of cells harbouring EV from each subpopulation was determined by an infectious center assay.

Electron microscopy, plaque assay and preliminary serological characterization of three ectromelia virus strains isolated in Poland in the period 1986–1988

SummaryThree strains of viruses have been identified as ectromedia virus (EV) based on their origin, clinical features, morphology, size of virions (140×220nm), replicative ability and specific cytoplasmatic fluorescence. The mean diameter of plaques produced by EV strains was 0.76mm (range 0.5–1.0mm). The neutralizing properties of the tested sera were evaluated by seroneutralization (SN) and hemagglutination inhibition (HAI) tests.

Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus‐induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host—the mouse—surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus–host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus‐induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell‐to‐cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin‐based cellular protrusions: short (actin tails and “dendrites”) and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton‐based cellular structures.

Mannosylated lipoarabinomannan balances apoptosis and inflammatory state in mycobacteria-infected and uninfected bystander macrophages.

To assess the role of mannosylated lipoarabinomannan (ManLAM) in the inflammatory and apoptotic response of mycobacteria-infected and uninfected, bystander cells we applied a mouse macrophage model of infection with avirulent strains--Mycobacterium bovis BCG, Mycobacterium tuberculosis (MTB) H37Ra and compared with a virulent MTB H37Rv strain infection. ManLAM contributed to the infection of macrophages by protection from apoptosis with stabilized Bcl-2 expression and down-regulated Bax expression for infected cells (BCG) or with stabilized Bcl-2 expression for uninfected bystander target cells (H37Ra). Additionally, ManLAM up-regulated FasL expression on the infected cells. Active extracellular signal-regulated kinase (ERK1/2) in BCG and H37Rv infection provided an anti-apoptotic effect by stabilization of anti-apoptotic Bcl-2 expression in the infected cells. Inhibitors specific for c-Jun-NH2-terminal kinase or stress-activated kinase (JNK) and p38 kinase decreased apoptosis of infected cells (BCG, H37Ra) and of uninfected bystanders (H37Ra) by down-regulating Bax. ManLAM significantly down-regulated production of pro-inflammatory IL-12 and TNF-alpha and activation of JNK by both avirulent strains. We conclude that by stabilization of Bcl-2 expression, down-regulation of JNK activity and down-regulation of pro-inflammatory cytokines production ManLAM can contribute to suppression of apoptosis and inflammatory reaction of uninfected, bystander cells.