In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes.

Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations- 1 μM (10(-3) mol/m(3)), 10 μM or 100 μM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD). The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA]) was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance-expansion of Treg cells and lymphocyte apoptosis-was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog

Characteristics of monoclonal antibodies used in immunofluorescense staining of lymphocyte antigens.

<p>Characteristics of monoclonal antibodies used in immunofluorescense staining of lymphocyte antigens.</p

ECTV infection does not trigger phenotypic maturation and inhibits LPS-induced maturation of GM-BM.

<p>Mock-, uvi-ECTV- or ECTV-infected GM-BM were left untreated or were treated with LPS for 24 h. Representative histograms showing the major histocompatibility complex (MHC) (A) or co-stimulatory (B) molecules expression on GM-BM. Numbers represent the MFI value and/or the percentage of positive cells for a given marker. Graphs show mean ± SD of MFI and/or percentage for indicated marker from at least three independent experiments (paired Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01). Statistical comparisons were between mock- or uvi-ECTV-treated DCs and ECTV-exposed DCs and between LPS- or uvi-ECTV+LPS-treated DCs and ECTV + LPS-exposed DCs. IsCon–isotype control.</p

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

<div><p>Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF–derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4⁺ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.</p></div

ECTV infection inhibits nuclear translocation of NF-κB, IRF3 and IRF7 in GM-BM.

<p>Indirect immunofluorescence analysis of nuclear translocation of NF-κB (A), IRF3 (B) and IRF7 (C) in mock-, uvi-ECTV- or ECTV-infected GM-BM untreated or treated with LPS for 24 h. Left panels show images of red fluorescence channel for the indicated transcription factor, right panel show images of merge fluorescence channels for the indicated transcription factor (red), viral antigen (green), and nuclear and viral DNA (blue). Arrowheads show viral factories (A, C) or vacuoles (B) in infected cells. Scale bars = 10 μm. Nuclear [N]: cytoplasmic [C] ratios of NF-κB (D), IRF3 (E) and IRF7 (F) were determined at single cell level by measuring of fluorescence signal intensities within the nucleus (stained with Hoechst 33342) and the cytosol. Analysis was performed on 50 cells/condition and experiment (from three independent experiments). Black lines indicate the mean values of each data set (Student’s <i>t</i>-test;^**<i>P</i>< 0.01).</p

Kinetics of ECTV replication in GM-BM.

<p>GM-BM were cultured in medium only (mock), medium containing UV-inactivated ECTV (uvi-ECTV) or live-ECTV (ECTV) for 4, 12 and/or 24 h. In some experiments cells were left untreated or were additionally treated with LPS (1 μg/ml). (A) Representative images of mock–, uvi-ECTV–and ECTV–treated GM-BM at 4 and 24 hpi stained with Hoechst 33342 (blue fluorescence) and pAbs anti-ECTV (green fluorescence). The magnified images are of the boxed regions. Arrows indicate viral particles; arrowheads show viral factories. Scale bars = 10 μm. (B) Representative histograms showing the percentage of ECTV⁺ cells at 4, 12 and 24 hpi of GM-BM. Numbers represent the percentage of ECTV⁺ cells. (C) The mean percentage of ECTV⁺ cells during infection in GM-BM. Error bars represent ± SD from three independent experiments.(D) Scanning electron microscopy micrograph of GM-BM surface at 24 hpi. The magnified images are of the boxed regions. Arrows indicate viral particles. Scale bars = 2.5 μm. IsCon–isotype control.</p

ECTV infection modulates the expression of genes engaged in the innate antiviral immune response.

<p>Log2 fold change of mRNA expression for genes associated with Toll-like receptor (TLR) (A), RIG I-like receptor (RLR) (B), IFN type I (C) and NOD-like receptor (NLR) (D) signaling. Average threshold cycle (C_T) values from PCR reactions were normalized against the average C_T values for the endogenous control <i>Atg12</i> from the same cDNA sample and shown as 2^(-ΔΔCT) were ΔC_T = C_{T gene}− C_{T atg12}. Graph columns represent the mean values of log2 fold change in mRNA expression from two independent experiments (Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01).</p

ECTV induces morphological changes and apoptosis in infected GM-BM.

<p>(A) Morphological changes of GM-BM infected with ECTV. GM-BM were cultured in medium only (mock), medium containing UV-inactivated ECTV (uvi-ECTV) or live-ECTV (ECTV) and were stimulated with or without LPS (1 μg/ml) for 24 h. Left panels show representative images of May-Grünwald-Giemsa stained GM-BM. Right panels demonstrate representative scanning electron microscopy micrographs of GM-BM. Scale bars = 5 μm. (B) The mean percentage of early and late apoptotic cells in GM-BM at 4, 12 and 24 hpi. Error bars represent ± SD from three independent experiments (Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01).</p

ECTV infection does not trigger phenotypic maturation and inhibits LPS-induced maturation of GM-BM.

<p>Mock-, uvi-ECTV- or ECTV-infected GM-BM were left untreated or were treated with LPS for 24 h. Representative histograms showing the major histocompatibility complex (MHC) (A) or co-stimulatory (B) molecules expression on GM-BM. Numbers represent the MFI value and/or the percentage of positive cells for a given marker. Graphs show mean ± SD of MFI and/or percentage for indicated marker from at least three independent experiments (paired Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01). Statistical comparisons were between mock- or uvi-ECTV-treated DCs and ECTV-exposed DCs and between LPS- or uvi-ECTV+LPS-treated DCs and ECTV + LPS-exposed DCs. IsCon–isotype control.</p