Human Cytomegalovirus Can Infect Dendritic Cell And Inhibit Its Function

The human cytomegalovirus (HCMV), a member of the beta-herpesvirinae subfamily, is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. It may, however, reactivate and cause severe disease in the absence of an effective immune response, as in immunologically immature and immunocompromised individuals, such as organ allografting, malignant, and human immunodeficiency virus (HIV)-infected patients.Dendritic cells (DCs) are important functional antigen-presenting cells that play an essential role in initiating and modulating immune responses.The maturation and subtypes of DC determine the balance of immune activation and tolerance .Their mature states are crucial for the induction of T-cell-mediated immune reactions.It was reported that HCMV could infect some kinds of cells,such as epithelial, endothelial, fibroblast, monocyte/macrophage cells,and so on.The main purpose of this research is to explore the efficiency of monocyte-derived DCs by HCMV, its effects on infected cells and the relationship with immune evasion. So our studies were initiated to address the following questions: (1) Can HCMV infect DCs? (2) The abnormal function of infected DCs,including immunological phenotypes, capacity of endocytosis and allogeneic stimulating of DCs. (3) What are the possible molecular mechanisms of these changes in infected DCs?In part 1, The results of the optical and transmission electron microscope revealed that the peripheral monocyte progenitors successfully developed into DCs with the combination of cytokine, GM-CSF, IL-4 and lipopolysaccharide in our culture system of DCs. The immature DCs, appeared to have much relatively shorter processes on the surface and fewer organells in the cytoplasmcompared with its counterpart-mature DCs,which turned to have even more abundant cytoplasmic extensions, more mitochondrias and less lysosomes,irregular large nuclei in the cytoplasm.DCs were infected by 50 TCIDJ0 HCMV.The infection efficiency was determined by varius methods. HCMV particles could be seen in nucleus and cytoplasma of infected DCs by electrion microscope. The relative expression of IE mRNA detected by RT-PCR in mature dendritic cells was lower than immature DCs at 12h after infecion, 0.102 ±0.020 and 0.862 + 0.124,respectively (PO.05). HCMV EA antigen was analyzed by indirect immunofluorescence assay after infection.lt could be found in (62.32+14.20)% immature DCs and (10.78 + 3.04)% mature DCs at 24h, repectively (PO.05). pp65 positive cells in immature DCs and mature DCs at 72h were (4.24 + 0.38)% and (0.82+0.13)%.It can be concluded from our experiments that HCMV can efficiently infect immature DCs. In part 2, To explore the influence of HCMV on DCs function, immunological phenorypes, capacity of endocytosis and allogeneic stimulating of infected DCs were measured.By using flow cytometry.an impaired ability of HCMV infected DCs to mature in response to LPS could be observed. Both HLA-ABCand HLA-DR molecules on the surface of DCs were significantly downregulated after inducing maturation by LPS compared with uninfected cells,(92.0 + 3.9)% versus (46.4 + 5.0)% for HLA-ABC molecule and (91.8 + 5.3)% versus (54.3 + 5.7)% for HLA-DR molecule(both PO.05). In contrast, the costimulatory molecules CD80.CD86 were slightly upregulated,(62.4 + 8.3)% versus (73.8 + 7.4)% for CD80 and (67.3+7.6)% versus (77.8 + 8.9)% for CD86 (P>0.05). CD83 was significantly upregulated to (30.2 + 7.4) %,which was similar to untreated immature DCs.Mean fluorescence intensity (MFI) measured by flow cytometry were used to detect the endocytosis capacity of DCs. HCMV infected immature DCs were significantly impaired in their ability to endocytose FITC-labeled dextran particles, 30-40% compared with untreated immature DCs.HCMV infected immature DCs showed depressed antigen presentation even after stimulated with maturation signal factor LPS (P0.05,both),which is similar with immature DCs.HCMV infected mature DCs had weaker stimulating capacity only when the ratio of DCs and T cells was 1:1 compared with untreated cells(P0.05).No significant difference was observed when the ratios were 1:10,1:50 and l:100,respectively.In part 3, to investigate possible molecular mechanisms underling these changes,actin microfilament cytoskeleton was studied in infected DCs.Actin microfilament cytoskeleton plays an important role in keeping normal function of DCs.Immature DCs can not differiate to mature stage after stimulated with maturation signal factor LPS when they were pretreated with LatB(0.1 u g/ml) or CytoD(2 P g/ml). HLA-ABC% CD80, CD86 and CD83 were downregulated compared with untreated mature DCs.No significant difference was observed for HLA-DR molecular.At 20,40,60,80min ,MFI of FITC-dextran in DCs treated with actin polymerization inhibitor LatB(0.1 u g/ml) were 65.4 + 10.2> 66.4 + 5.7, 63.2±12.6> 70.5 + 8.9,respectively.They were obviously lower than untreated DCs.Actin depolymerization inhibitor CytoD(2 u g/ml) had similar results.HCMV AD 169 was propagated in human fibroblasts (HF). It was found that HF cells infected by HCMV turned from thin shuttle shape to round and thick ball shape, even escaping from wall. Cell shape changed more dominantly as the HCMV titer increased. Cytoskeleton alteration was determined by microfilament fluorescence probe. In infected HF cells, microfilaments were ruptured, arranged turbulently. cells merged.The fluorescence density of microfilament obviously reduced.The ratio of P-actin / GAPDH detected by RT-PCR in untreated DCs is 2.302+0.427. It increased to 7.025 + 0.873 at 24h after infection(P<0.05). The expression of 0-actin mRNA decreased 1.528+0.204 and 0.432+0.080 at 72h,96h after infection,respectively.Compared with uninfected DCs.the expression of 3 -actin protein decreased to 90.1%,73.8% and 34.2 % respectively at 24h,72h and 96h after infection. There is no significant difference of the expression of P-tubulin protein.The proliferative T cell response was drastically reduced, however, this reduced capacity to stimulate alloreactive T cells by HCMV infected DCs can be partly restored by adding actin polymerization activator PMA(25nM). Similar results could be found for immunological phenotypes and endocytosis capacity of infected DCs.We propose that the infection of immature DCs allows HCMV to evade immune recognition and T cells attack. Actin cytoskeleton abnormity in infected cells may play a pivotal role in HCMV triggered immunosuppression.The reduced capacity to stimulate alloreactive T cells of HCMV infected DCs is possible due tothe effect of actin microfilament of HCMV on DCs, which inhibit the immmture DCs develop to mature DCs.During bone marrow transplantation in clinical work,the active infection of HCMV on DCs may deteriorate the attack of DCs on tumour cells.Further research on the mechanism of HCMV infection and immune evasion of HCMV.In summary, some conclusions can be drawn from our results.(l) HCMV can efficiently infect DCs,especially immature DCs. (2) HCMV can inhibit the antigen presentation capacity of DCs. (3) Actin cytoskeleton may play an importan role in keeping DCs function. (4) HCMV induces actin cytoskeleton abnormal in infected DCs. (5) Actin polymerization activator PMA can partly restored the antigen presentation capacity of HCMV infected DCs.