Quantification of T-cell dynamics during latent cytomegalovirus infection in humans

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8⁺ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8⁺ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8⁺ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8⁺ T-cell populations. The lifespans of circulating CMV-specific CD8⁺ T-cells did not differ significantly from those of bulk memory CD8⁺ T-cells, and the lifespans of bulk memory CD8⁺ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4⁺ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8⁺ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8⁺ T-cells are not due to longer lifespans of these cells.     

Murine cytomegalovirus infection directs macrophage differentiation into a pro-inflammatory immune phenotype: implications for atherogenesis

We have previously demonstrated that mouse cytomegalovirus (MCMV) aggravates atherosclerosis in apolipoprotein E knockout (apoE(-/-)) mice, most likely by enhancing both systemic and local (e.g. in the vascular wall) cytokine production. However, until now it was unclear which cell type is responsible for this enhanced pro-inflammatory cytokine production. In this study we focused on the macrophage (mPhi), which besides being an important source of such cytokines, is known to be an important player in both atherosclerosis and viral clearance. We investigated whether MCMV could induce a pro-inflammatory immune mPhi phenotype, which ultimately may contribute to the development of atherosclerosis. To this end, peritoneal exudate cells (PEC) were elicited in apoE(-/-) mice by either MCMV or thioglycolate injection, and mPhi were phenotyped at 1 week post-intraperitoneal injection. MCMV-induced peritoneal mPhi contained MCMV DNA but had limited MCMV mRNA expression, indicating latent infection. These mPhi showed increased production of interferon-gamma (IFNgamma), exclusive production of interleukin-18 (IL-18) and increased expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86, when compared with thioglycolate-induced mPhi. From these results, we conclude that intraperitoneal injection of MCMV induces an immune-responsive exudate in which at 7 days post-infection, MCMV-infected mPhi express a pro-inflammatory immune phenotype. As such, the MCMV-induced mPhi may be an important player in aggravating atherosclerosis through systemic and/or local immune activation.     

Effect of anti-mouse macrophage serum on murine cytomegalovirus infection.

Rabbit anti-mouse macrophage serum (AMS) was used to study the role played by macrophages against murine cytomegalovirus infection. Treatment of mice with AMS enhanced morbidity and mortality following virus infection. These results are discussed in relation to the role of macrophages against virus infections.     

Autophagy as a macrophage response to bacterial infection

The macrophage is a key component of host defense mechanisms against pathogens. In addition to the phagocytosis of bacteria and secretion of proinflammatory mediators by macrophages, autophagy, a process involved in turnover of cellular material, is a recently identified component of the immune response to bacterial infection. Despite the bactericidal effect of autophagy, some species of intracellular bacteria are able to survive by using one or more strategies to avoid host autophagic attack. Here, we review the latest findings on the interactions between bacteria and autophagy in macrophages. ? 2012 IUBMB Life, 64(9): 740-747, 2012.     

Viral interleukin-10 expressed by human cytomegalovirus during the latent phase of infection modulates latently infected myeloid cell differentiation

The human cytomegalovirus UL111A gene is expressed during latent and productive infections, and it codes for homologs of interleukin-10 (IL-10). We examined whether viral IL-10 expressed during latency altered differentiation of latently infected myeloid progenitors. In comparison to infection with parental virus or mock infection, latent infection with a virus in which the gene encoding viral IL-10 has been deleted upregulated cytokines associated with dendritic cell (DC) formation and increased the proportion of myeloid DCs. These data demonstrate that viral IL-10 restricts the ability of latently infected myeloid progenitors to differentiate into DCs and identifies an immunomodulatory role for viral IL-10 which may limit the host's ability to clear latent virus.     

Splenic hematoma as a first manifestation of cytomegalovirus infection

Splenic rupture is rare but life threatening complication of mononucleosis syndrome. It has been suggested that subcapsular splenic hematoma formation precedes rupture. The case of 44-year-old, previously healthy, male with splenic hematoma occurring after rising of heavy cargo is reported. Mononucleosis syndrome was suggested based on routine laboratory tests (elevated white blood cell count with predominance of lymphocytes and raised serum transaminases) and CMV infection was confirmed by serological test. Nonoperative management was used since the patient was hemodynamically stable with no further signs of splenic rupture. The same approach has been used in growing number of cases of patients with spontaneous splenic rupture in mononucleosis syndrome. Importance of considering splenic hematoma and/or rupture if abdominal pain occurs in the course of mononucleosis syndrome is outlined as well as importance of routine laboratory tests in suspecting mononucleosis syndrome in otherwise clinically silent patient.     

Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.     

Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections.

Spleen cells which replicate murine cytomegalovirus (MCMV) during acute infection in vivo were identified by electron microscopy and combined immunocytochemical staining and in situ cytohybridization. Most infected cells, as defined by in situ hybridization for viral RNA with MCMV-specific probes, were shown to be positive for factor VIII-related antigen and negative for Ia, Thy-1, and F4/80 antigens. Electron microscopic ultrastructural observations indicated that the infected cells in the spleen are predominantly sinusoidal-lining cells. We also studied reactivation of MCMV from latently infected mice by cocultivation of spleen cells with mouse embryo fibroblasts. Virus was only recovered from cells in preparations of stromal (or reticular) fragments, and not from spleen cell suspensions. Neither removal of immunoglobulin-bearing cells from the stromal fragments by panning nor depletion of Thy-1- and Ia-bearing stromal cells by treatment with monoclonal antibodies and complement reduced the frequency of reactivation of MCMV. These data suggest that T lymphocytes, mature B lymphocytes, and other Ia-bearing cells are not predominant reservoirs of latent MCMV.     

Permissive human cytomegalovirus infection of a first trimester extravillous cytotrophoblast cell line

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy.     

Leishmania amazonensis: xylitol as inhibitor of macrophage infection and stimulator of macrophage nitric oxide production

Xylitol is a sugar alcohol being explored for clinical uses. The aim was to evaluate the effects of xylitol on Leishmania amazonensis-infected J774A.1 macrophages. Macrophages were infected with L. amazonensis for 3h, washed and incubated with 2.5 or 5.0% xylitol for 24, 48, and 72 h at 37 degrees C. Infection indexes for macrophages incubated only in medium were compared to those treated with xylitol. Cell viability and nitric oxide production were determined each time. Xylitol did not affect L. amazonensis or J774A.1 cell viabilities. Xylitol at 5.0% stimulated nitric oxide production by macrophages at 72 h (p<0.01). At 2.5 and 5.0%, xylitol inhibited nitric oxide production by L. amazonensis at 48 h (p<0.05) when compared to control. Infection indexes were significantly lower at 72 h (p<0.05), (16.9% and 9.6%) in cells cultivated with 2.5 and 5.0% xylitol, respectively, compared to control (38.4%). Results suggest a potential leishmanicidal action of the xylitol on infected macrophages.     

Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.     

The placenta as a site of cytomegalovirus infection in guinea pigs.

The development of cytomegalovirus (CMV) infection in the placenta was studied in Hartley guinea pigs inoculated at midgestation, and its role in determining the outcome of fetal CMV infection was assessed. A hematogenous spread of CMV from the mother to the placenta occurred early during the course of the infection. However, the virus remained present in placental tissues long after CMV had been cleared from maternal blood (i.e., 3 and 4 weeks postinoculation). At that time, the virus was able to replicate in placental tissues in the presence of specific maternal antibodies. Viral nucleocapsids were seen within nuclei of trophoblastic cells, and virions were present surrounding infected cells. In addition, typical CMV-induced histopathological lesions bearing CMV antigens were consistently localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobium. Whenever CMV infection of the fetus occurred, virus was isolated from the associated placenta. Among placental-fetal units with CMV-infected placentas, only 27% of the fetuses were found to be infected. In addition, there was a delay in the establishment of the infection in the fetus in relation to the placenta, although frequencies of virus isolation in placental and fetal tissues peaked at 3 weeks after CMV inoculation. These results suggest that during primary CMV infection of pregnant guinea pigs, the placenta not only serves as a reservoir for CMV but also acts to limit transmission of the virus to the fetus.     

Genetic labeling reveals altered turnover and stability of innate lymphocytes in latent mouse cytomegalovirus infection

Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B-eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H(+) and Ly49H(-) NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were "legacy" (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.