Characterisation of prrsv1 infection of bone marrow-derived dendritic cells

Resumen

The present thesis aims to characterize the attachment, replication and the induction of apoptosis during PRRSV infection in bone marrow-derived dendritic cells (BMDC), including both immature (i) and matue (m) BMDC. Three PRRSV1 isolates (3249, 3262 and 3267) were used. The kinetics of replication were assessed by titrating cell culture supernatants in macrophages. The viral yield in iBMDC at 12 and 24 hpi was significantly higher than in mBMDC, and the replication of two isoaltes (3249 and 3262) peaked earlier in iBMDC (24 hpi) compared to in mBMDC (48hpi). These indicated that iBMDC were more efficient than mBMDC in supporting viral replication. This feature was not related to the proportion of CD163+ cells nor the levels of IFN-α in the cultures. In addition, the replication efficiency was strain-dependent. Isolate 3262 showed the lowest titres in both cell types at 12, 24 and 48 hpi, consistent with the lowest infected proportion by 3262 in flow cytometry. This is compatible with the lowest binding rate of this isolate (confocal microscopy). The attachment and replication was further studied in association with the expression of three receptors, PoSn, CD163 and heparan sulphate. A three-colour confocal microscopy staining (PoSn, CD163 and PRRSV) on iBMDC showed that attachment occurred on the four subsets defined by PoSn and CD163, which was not fully avoided by removal of heparan sulphate from the cell surface. This indicated the attachment of PRRSV1 on BMDC may occur beyond the intervention of heparan sulphate, PoSn and CD163. For examining replication with regards to these receptors, a two-colour confocal microscopy staining (CD163/PRRSV or PoSn/PRRSV) was performed. Replication was observed in apparently PoSn- and CD163- cells. As CD163 is the only recognized essential receptor for PRRSV, its expression together with the infection by isolate 3267 on iBMDC was further examined by flow cytometry. As a result, 8.4 ± 0.5% of apparently CD163- cells were labelled as infected. To further clarify this, a sorting experiment was designed based on the variable amounts of CD163 (CD163-, CD163lo and CD163hi). The first sorting focused on “beyond doubt” CD163- cells. The second sorting grouped CD163- cells together with CD163lo. Unsorted iBMDC were used as controls. In consequence, the “beyond doubt” CD163- cells were not infected by 40 hpi. When CD163- were sorted with CD163lo, the infected CD163- population (0.6 ± 0.07%) appeared again by 40 hpi and when the incubation was extended to 60 hpi, the percentage increased more (1.6% ± 0.08%). Consistently, the proportion of infected cells at 60 hpi was higher than the initial number of CD163+ cells. These can be explained by the generation of new CD163lo beyond the cytometer’s sensitivity, or the milieu created by CD163+ infected cells resulted in that CD163- susceptible population, or the yet unknown receptors developed. Regarding the induction of apoptosis, positive signals for cleaved caspase-3 in PAM, were observed in both infected and bystander cells for all three isolates (confocal microscopy), while in BMDC they were mainly in bystanders, indicative of different apoptosis triggering pathways in PAM and BMDC. Besides, at m.o.i. 0.1, the caspase-3 signal in BMDC peaked later (48 hpi) than in PAM (24 hpi), which may allow more cycles of viral replication and result in higher viral yields in BMDC. Further examination of inoculated BMDC cultures for apoptosis/necrosis showed significant differences between isolates with apparently induced apoptosis/necrosis of 3249 and 3267 but rarely of 3262. Neutralization of IL-10 induced by 3262 resulted in the occurrence of apoptotic cells, but this did not happen with a second IL-10-induced isolate (2988). The above-mentioned results will be useful to understand the role of DC in PRRSV pathogenesis.