Indoleamine 2,3-Dioxygenase Is Involved in Defense against Neospora caninum in Human and Bovine Cells ▿

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii. In nature this parasite is found especially in dogs and cattle, but it may also infect other livestock. The growth of N. caninum, which is an obligate intracellular parasite, is controlled mainly by the cell-mediated immune response. During infection the cytokine gamma interferon (IFN-γ) plays a prominent role in regulating the growth of N. caninum in natural and experimental disease. The present study showed that induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) is responsible for the inhibition of parasite growth that is mediated by IFN-γ-activated bovine fibroblasts and endothelial cells. This antiparasite effect could be abrogated by addition of tryptophan, as well as by the IDO-specific inhibitor 1-l-methyltryptophan. In conclusion, our data show that human and bovine cells use the same effector mechanism to control the growth of N. caninum

Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

Human mesenchymal stromal cells (MSC) possess immunosuppressive and antimicrobial effects that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Therefore MSC represent a promising novel cellular immunosuppressant which has the potential to control steroid-refractory acute graft versus host disease (GvHD). In addition, MSC are capable of reducing the risk of infection in patients after haematopoietic stem cell transplantation (HST). Recent data indicate that signals from the microenvironment including those from microbes may modulate MSC effector functions. As Cytomegalovirus (CMV) represents a prominent pathogen in immunocompromised hosts, especially in patients following HST, we investigated the impact of CMV infection on MSC-mediated effects on the immune system. We demonstrate that CMV-infected MSC lose their cytokine-induced immunosuppressive capacity and are no longer able to restrict microbial growth. IDO expression is substantially impaired following CMV infection of MSC and this interaction critically depends on intact virus and the number of MSC as well as the viral load. Since overt CMV infection may undermine the clinical efficacy of MSC in the treatment of GvHD in transplant patients, we recommend that patients scheduled for MSC therapy should undergo thorough evaluation for an active CMV infection and receive CMV-directed antiviral therapy prior to the administration of MSC

HCMV blocks IDO activity and subsequent antibacterial effects observed in co-cultures of activated T cells and HFF.

<p>Peripheral blood mononuclear cells (PBMC; 1×10⁵/well), stimulated with a CD3-directed mAb (OKT3), were co-cultured with HFF (2×10⁴/well) in the absence or presence of HCMV (MOI 5). As control UV-inactivated HCMV (uvCMV) was used. (A) After three days IDO activity was determined and is presented as mean kynurenine production +/− SEM of 4 independent experiments, each done in triplicates. The OD measured in the negative control (unstimulated HFF) is subtracted in all groups. (B) HFF/PBMC co-cultures were set up as described above. After three days cultures were infected with <i>S. aureus</i> (10–100 cfu/ml) and bacterial growth was determined photometrically 24 hours later. As a control L-tryptophan (100 µg/ml) was added at the time point of bacterial infection. Data are given as mean OD_{(620 nm)} +/− SEM of three experiments, each done in triplicates. Significant differences (p<0.05) as compared to the positive control are marked by asterisks.</p

HCMV infection abrogates IFN-γ-induced IDO activity and subsequent antimicrobial effects.

<p>(A) HFF (3×10⁴/well) were stimulated with different amounts of IFN-γ in cell culture medium containing 0.6 mM tryptophan. The cultures were infected with HCMV (MOI 5) at the time point of IFN-γ stimulation, after 24 hours or 48 hour, respectively. After three days the kynurenine production by the cells was determined to directly measure IDO enzyme activity using Ehrlich’s reagent. Data are given as mean kynurenine production +/− SEM of five independent experiments, each done in triplicates. The OD measured in the negative control (unstimulated HFF) is subtracted in all groups. (B) HFF were stimulated with IFN-γ and infected with HCMV as described above. After three days cultures were infected with <i>S. aureus</i> (10–100 cfu/well) and bacterial growth was determined photometrically 24 hours later. Data are given as mean optical density +/− SEM of three independent experiments, each done in triplicates. (C) HFF were stimulated with IFN-γ and infected with HCMV as described above. Three days after activation the cells were infected with <i>T. gondii</i> (2×10⁴/well) and parasite growth was determined using [³H]-uracil three days later. Data are given as mean cpm +/− SEM of four independent experiments, each done in triplicates. HCMV added within the first 24 h of culture significantly reduced (p<0.05) IFN-γ-induced IDO activity and subsequent antibacterial and antiparasitic effects. (D) HFF cultures were co-infected with <i>T. gondii</i> and HCMV. After two days an immunofluorescence analysis was performed. While <i>T. gondii</i> parasites were detected with an anti-GRA9 antibody stained in green within the parasitophorous vacuole, HCMV was detected with an anti-HCMV-pp72 antibody stained in red [panel a)] within the nucleus of the host cell. As a control panel b) shows DAPI nuclear staining in blue with merged phase contrast.</p

HCMV infection abrogates the immunosuppressive effect of human fibroblasts

<p>. (A) PBMC (2×10⁵/well) were activated with a CD3-directed mAb (OKT3) and cultured in the presence or absence of HFF which were infected with HCMV (MOI 5) or not. After three days T cell proliferation was assessed using [³H]-thymidin. Data are given as mean cpm +/− SD of a representative experiment performed in triplicates. As a control 1- MT was used. (B) Different amounts of HFF, either HCMV infected (MOI 5) or not, were co-cultured with OKT3-activated PBMC. Thereafter T cell proliferation was determined as described above. Data are given as % of positive control without HFF. Each dot represents a single experiment (n = 7), each performed in triplicates. A significant inhibition of T cell proliferation by HFF is marked with asterisks.</p