The in Vitro Inhibitory Effect of Ectromelia Virus Infection on Innate and Adaptive Immune Properties of GM-CSF-Derived Bone Marrow Cells Is Mouse Strain-Independent

Abstract

Ectromelia virus (ECTV) belongs to the Orthopoxvirus genus of the Poxviridae family and is a natural pathogen of mice. Certain strains of mice are highly susceptible to ECTV infection and develop mousepox, a lethal disease similar to smallpox of humans caused by variola virus. Currently, the mousepox model is one of the available small animal models for investigating pathogenesis of generalized viral infections. Resistance and susceptibility to ECTV infection in mice are controlled by many genetic factors and are associated with multiple mechanisms of immune response, including preferential polarization of T helper (Th) immune response toward Th1 (protective) or Th2 (non-protective) profile. We hypothesized that viral-induced inhibitory effects on immune properties of conventional dendritic cells (cDCs) are more pronounced in ECTV-susceptible than in resistant mouse strains. To this extent, we confronted the cDCs from resistant (C57BL/6) and susceptible (BALB/c) mice with ECTV, regarding their reactivity and potential to drive T cell responses following infection. Our results showed that in vitro infection of granulocyte-macrophage colony-stimulating factor-derived bone marrow cells (GM-BM—comprised of cDCs and macrophages) from C57BL/6 and BALB/c mice similarly down-regulated multiple genes engaged in DC innate and adaptive immune functions, including antigen uptake, processing and presentation, chemokines and cytokines synthesis, and signal transduction. On the contrary, ECTV infection up-regulated Il10 in GM-BM derived from both strains of mice. Moreover, ECTV similarly inhibited surface expression of major histocompatibility complex and costimulatory molecules on GM-BM, explaining the inability of the cells to attain full maturation after Toll-like receptor (TLR)4 agonist treatment. Additionally, cells from both strains of mice failed to produce cytokines and chemokines engaged in T cell priming and Th1/Th2 polarization after TLR4 stimulation. These data strongly suggest that in vitro modulation of GM-BM innate and adaptive immune functions by ECTV occurs irrespective of whether the mouse strain is susceptible or resistant to infection. Moreover, ECTV limits the GM-BM (including cDCs) capacity to stimulate protective Th1 immune response. We cannot exclude that this may be an important factor in the generation of non-protective Th2 immune response in susceptible BALB/c mice in vivo.

In Vivo Characterization of a Bank Vole-Derived Cowpox Virus Isolate in Natural Hosts and the Rat Model

“…The basis of resistance or susceptibility of a mammal species for CPXV infection is determined by host characteristics [41,42] as well as viral factors [43]. An example is the outcome of ECTV infection in laboratory mice: resistance and susceptibility are controlled by genetic factors of the host and are associated with multiple immune response mechanisms [44]. However even C57/BL6 mice, resistant to infection, respond serologically to ECTV inoculation [45].…”

Ectromelia virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α-tubulin acetylation

“…Our experiments were conducted on granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow cells (GM-BM) composed of conventional DCs (cDCs) and macrophages. GM-BM cells were generated from bone marrow precursors obtained from femurs and tibias of BALB/c mice with ethical approval as described previously [ 14 , 15 ]. After 8 days of culture, cDCs were enriched by magnetic-activated cell sorting (MACS) using CD11c + -labeled magnetic beads (Miltenyi Biotec) and the surface expression of CD11c, CD11b, MHC II and CD205 molecules was assessed by flow cytometry as reported previously [ 14 , 15 ].…”

“…GM-BM cells were generated from bone marrow precursors obtained from femurs and tibias of BALB/c mice with ethical approval as described previously [ 14 , 15 ]. After 8 days of culture, cDCs were enriched by magnetic-activated cell sorting (MACS) using CD11c + -labeled magnetic beads (Miltenyi Biotec) and the surface expression of CD11c, CD11b, MHC II and CD205 molecules was assessed by flow cytometry as reported previously [ 14 , 15 ]. CD11c-enriched GM-BM cells were left uninfected (mock) or were infected with ECTV (ATCC VR-1374) at a multiplicity of infection (MOI) of 1 and/or were treated with 1 µg of lipopolysaccharide (LPS; Escherichia coli 0111:B4; Sigma-Aldrich) per ml for 24 h.…”

“…Expression of tubulin- and actin-cytoskeleton-related genes was evaluated using quantitative real-time PCR (qPCR) analysis. RNA isolation and reverse transcription (RT) were performed as described previously [ 14 , 15 ]. Real-time PCR was performed using Real-Time Ready Custom Panel 96 pre-coated plates and LightCycler 480 Probes Master Mix (Roche Diagnostics) according to the manufacturer’s instructions.…”

“…Taken together, our data indicate that ECTV is able to stabilize MTs within immune cells, probably to facilitate the spread of the virus. Interestingly, our previous study showed that LPS treatment of ECTV-infected GM-BM cells derived from BALB/c and C57BL/6 mice increased both the percentage of ECTV + cells and the number of infectious virus particles at 24 hpi when compared to LPS-untreated ECTV-infected cells [ 15 ]. Therefore, it is highly likely that LPS-induced extensive acetylation of MTs in ECTV-infected cells may additionally increase the dissemination of the virus.…”