Cytomegalovirus infection aggravates atherogenesis in apoE knockout mice by both local and systemic immune activation

Since the 1970s, cytomegalovirus (CMV) infection has been associated with atherosclerotic disease. However, the exact contribution of the virus remains uncertain. In this article we describe both a direct and indirect immune-mediated effect of the virus on the disease process. Eight-week-old apolipoprotein E (apoE) knockout mice were infected with mouse CMV (MCMV) or mock injected, and they were sacrificed at 2 and 20 weeks post-injection (p.i.) to study atherosclerosis, vascular wall IFNgamma and TNFalpha expression and MCMV spread. To study plasma IFNgamma and TNFalpha levels, blood was collected at 1, 2, 4 and 6 days p.i. in addition to days of sacrifice. Plasma cytokine levels were increased after MCMV infection at early time points and decreased to mock levels at 2 and 20 weeks p.i. At 2 weeks p.i., more aortic arch samples showed local cytokine expression after MCMV infection. The number of early atherosclerotic lesions and the percentage of mice containing early lesions were increased at 2 weeks p.i., while at 20 weeks p.i., the MCMV-induced effect on atherogenesis was seen on the late lesions. In conclusion, MCMV infection induces a systemic immune response reflecting an indirect effect of MCMV infection on atherosclerosis in addition to a local aortic immune response reflecting a direct effect of the virus on the atherosclerotic process.     

Intraperitoneal immune cell responses to Eubacterium saphenum in mice

Oral asaccharolytic Eubacterium saphenum, which are newly isolated gram-positive rods and one of the predominant microorganisms in human periodontal pockets, were injected intraperitoneally in mice to elucidate their pathogenicity in periodontal diseases. Infiltrating immune cells in the peritoneal exudate were quantitated and intracellular T cell (CD4+/CD8+/gammadelta+) production of cytokines IL-4 and IFN-gamma which are related to cellular and humoral immunity, respectively, was determined. Neutrophils appeared first in peritoneal exudates, followed by macrophages and lymphocytes, after the injection of either E. saphenum or Porphyromonas gingivalis. Intracellular IL-4+ and IFN-gamma+ gammadelta T cells were detected in the exudates after the injection of E. saphenum (4.6 +/- 0.8% and 10.1 +/- 1.4%, respectively) and P. gingivalis (5.3 +/- 1.6% and 10.1 +/- 2.1%, respectively). The intracellular production of IL-4/IFN-gamma in CD4+/CD8+ T cells was rather low indicating that the main response was from gammadelta T cells which initiated the immune reactions in mouse peritoneal cavities after injection of E. saphenum or P. gingivalis. Serum IgG and IgM levels were elevated in animals injected with E. saphenum and similarly with P. gingivalis. The present study showed that with slight differences, similar modes of cell response and cytokine and Ig production were observed after intraperitoneal injection of both E. saphenum and P. gingivalis, indicating that E. saphenum may play just as important a role in periodontal diseases as P. gingivalis.     

Cutting edge: Overlapping functions of TLR7 and TLR9 for innate defense against a herpesvirus infection

As initially demonstrated with murine cytomegalovirus (MCMV), plasmacytoid dendritic cells (pDCs) are the major source of IFN-alpha/beta in response to a variety of viruses in vivo. However, contradictory results have been obtained pertaining to the mechanisms promoting IFN-alpha/beta production by pDCs in response to MCMV. In this study we show that TLR7 and TLR9 exert redundant functions for IFN-alpha/beta, IL-12p40, and TNF-alpha production by pDCs in vivo during MCMV infection. In contrast, we confirm that systemic production of IL-12p70 strictly depends on TLR9. The combined loss of TLR7 and TLR9 recapitulates critical features of the phenotype of MyD88-deficient mice, including a dramatic decrease in systemic IFN-alpha/beta levels, an increase in viral load, and increased susceptibility to MCMV-induced mortality. This is the first demonstration of the implication of TLR7 in the recognition of a DNA virus.     

Murine cytomegalovirus downregulates interleukin-17 in mice with retrovirus-induced immunosuppression that are susceptible to experimental cytomegalovirus retinitis

Interleukin-17 (IL-17), a pro-inflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by splenic CD4+ T cells. Based on additional studies using IL-10 ?/? mice infected systemically with MCMV and IL-10 ?/? mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10.     

Simultaneous Th1-type cytokine expression is a signature of peritoneal CD4+ lymphocytes responding to infection with Listeria monocytogenes

The robust murine response to infection with Listeria monocytogenes makes an excellent model to study the functional development of immune cells. We investigated the cellular immune response to i.p. infection using intracellular cytokine staining to identify Ag-specific lymphocytes. CD4(+) peritoneal exudate cells obtained 10 days postinfection predominantly coexpressed TNF-alpha, IFN-gamma, and IL-2 after polyclonal or Ag stimulation. A population of cells simultaneously making TNF-alpha and IFN-gamma was also detected but at a lower frequency. By following the kinetics of the response to Listeria, we found that CD4(+) lymphocytes coexpressing TNF-alpha and IFN-gamma dominated on day 6 postinfection and then declined. From days 10-27, TNF-alpha(+)IFN-gamma(+)IL-2(+) (triple-positive) was the most prevalent cytokine phenotype, and the frequency steadily declined. These characteristic cytokine expression patterns were observed in both primary and secondary responses to Listeria infection and developed even when infection was terminated with antibiotic treatment. A cytokine-assisted immunization procedure resulted in both double- and triple-positive cells, but the clear predominance of triple-positive cells required Listeria infection. Triple-positive cells were preferentially noted in the peritoneal cavity tissue site; spleen cells displayed a predominant population of double-positive T cells (TNF-alpha(+)IFN-gamma(+)). We speculate that the appearance of triple-positive cells represents a functionally significant subset important in host defense at nonlymphoid tissue sites.     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

Murine cytomegalovirus infection can enhance hybrid resistance through modulation of host natural killer activity

Studies were undertaken to assess the effect of murine cytomegalovirus (MCMV) in two different models involving injection of parental cells into F1 hosts. In both of these systems, MCMV-induced enhancement of hybrid resistance was found. In the first model, parent-into-F1 graft-vs-host reaction, MCMV infection of (C57BL/6 x C3H)F1 (B6C3F1) hosts was found to prevent the GVHR normally induced by injection of B6 parental splenocytes into the F1 hosts. The second model involved injection of parental bone marrow into lethally irradiated B6C3F1 and (C57BL/6 x DBA/2)F1 (B6D2F1) hosts. These irradiated hosts are known to exhibit resistance to engraftment by parental C57BL/6 (B6) bone marrow. This resistance was found to be markedly enhanced by injection of the hosts with MCMV 3 days before irradiation and bone marrow injection. In contrast, engraftment into B6C3F1 hosts of syngeneic marrow, or bone marrow from the C3H parent, was not affected by MCMV infection. Engraftment of DBA/2 marrow into B6D2F1 hosts was reduced at lower doses of injected marrow, suggesting enhanced resistance against the minor Hh Ag Hh-DBA. To test whether the MCMV-induced enhancement of resistance was mediated by NK cells, splenic NK activity (YAC-1 killing) and frequency (NK1.1 staining) were assessed. Both parameters were found to be elevated at 3 days after MCMV infection but to return to normal levels by 9 days. B6 bone marrow engraftment was in fact found to be normal when the marrow was administered to F1 mice 9 days after MCMV infection. Furthermore, anti-asialoGM1 administration prevented MCMV-induced enhancement of resistance to marrow engraftment. Thus, the NK enhancement resulting from MCMV infection appears to play a major role in the enhanced HR observed in the marrow engraftment model. This effect may be of importance in clinical bone marrow transplantation, a situation in which patients are susceptible to viral infection.     

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.     

A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8(+) T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8(+) T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8(+) T cell responses to MCMV - both conventional memory responses and those undergoing long-term expansion or "inflation". We infected LMP7(-/-) and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8(+) T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory "inflating" epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8(+) T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.     

CD4+CD25+ regulatory T cells control innate immune reactivity after injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury.     

Helper and suppressor functions of panned populations of immune peritoneal T cells with reactivity to the rat mammary adenocarcinoma 13762A

The phenotype of glass-adherence-depleted tumor-immune peritoneal exudate lymphocytes (PEL) which generated anti-tumor reactivity against the rat mammary adenocarcinoma 13762A in vitro was examined by indirect panning. Monoclonal antibodies W3/25 and OX8, directed against the CD4 and CD8 differentiation antigens, respectively, were used to separate immune PEL into subsets of functionally different T cells. The panned populations of immune PEL were examined for anti-tumor reactivity in three different in vitro assays. Tumor-specific proliferation, tumor-specific induction of the helper lymphokine interleukin 2 (IL-2), and tumor-specific induction of an antiproliferative tumor-induced suppressor lymphokine (TISL) were determined. Panning experiments together with indirect immunofluorescence analysis of unpanned immune PEL indirectly indicated that a proportion of cells (15-20%) coexpressed CD4 and CD8 antigens. Strong tumor-specific proliferation and IL-2 and TISL production were generated from these double-positive cells, as determined by double-panning experiments. Significant tumor-specific proliferation and IL-2 production were produced also from CD4+CD8- cells, but TISL production was minimal, and could be accounted for by contaminating CD4+CD8+ cells. CD4-CD8+ cells produced negligible responses against the tumors as measured by these three assays, and no synergistic responses were demonstrated when CD4-CD8+ cells were incubated together with CD4+CD8- cells. These data demonstrated that CD4+CD8+ T cells exist in primed populations of rat peripheral lymphocytes, and that both helper and suppressor functions were generated from these double-positive cells.     

Modulation by biologic response modifiers of hepatitis C virus antigen-independent cytokine secretion in blood mononuclear cells.

The systemic hepatitis C virus (HCV) antigen-non-specific cytokine responses were investigated in cultures of peripheral blood mononuclear cells. Cytokines of the T helper (Th) 1 [interferon (IFN)gamma and interleukin (IL)-2] and Th2 (IL-4 and IL-10) phenotype, and the pro-inflammatory cytokines tumour necrosis factor alphaIL-1beta and IL-6, were secreted by the cells activated by the HCV antigen-independent pathway. Furthermore, these cytokine responses were shifted towards a Th1 predominance by treatment with IFN-beta and with IL-2, whereas cytokine responses were selectively amplified towards a combined Th1-Th2 profile by granulocyte-macrophage- but not by macrophage colony-stimulating factor. Moreover, pro-inflammatory cytokine production was significantly enhanced by IFN-beta and augmented dose-dependently with GM-CSF and IL-2. Therefore, these immune mediators can promote unique-as well as overlapping-systemic cytokine responses that may be relevant for immunotherapeutic interventions to control HCV infection.     

Cutting edge: murine cytomegalovirus induces a polyfunctional CD4 T cell response

CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-A(b)-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-gamma, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-gamma production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen.     

Impaired hepatic insulin signalling in PON2-deficient mice: a novel role for the PON2/apoE axis on the macrophage inflammatory response

Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)(-/-) mice, relative to apoE(-/-) mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE(-/-) mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE(-/-) mice is directly associated with altered production of macrophage pro-inflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE(-/-) mice is mediated by a shift in the balance of NO and ONOO(-) (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.     

Flipping the switches: CD40 and CD45 modulation of microglial activation states in HIV associated dementia (HAD)

Microglial dysfunction is associated with the pathogenesis and progression of a number of neurodegenerative disorders including HIV associated dementia (HAD). HIV promotion of an M1 antigen presenting cell (APC) - like microglial phenotype, through the promotion of CD40 activity, may impair endogenous mechanisms important for amyloid- beta (Aβ) protein clearance. Further, a chronic pro-inflammatory cycle is established in this manner. CD45 is a protein tyrosine phosphatase receptor which negatively regulates CD40L-CD40-induced microglial M1 activation; an effect leading to the promotion of an M2 phenotype better suited to phagocytose and clear Aβ. Moreover, this CD45 mediated activation state appears to dampen harmful cytokine production. As such, this property of microglial CD45 as a regulatory "off switch" for a CD40-promoted M1, APC-type microglia activation phenotype may represent a critical therapeutic target for the prevention and treatment of neurodegeneration, as well as microglial dysfunction, found in patients with HAD.     

Dietary gamma-linolenic acid dose-dependently modifies fatty acid composition and immune parameters in rats.

gamma-linolenic acid (GLA) has been reported to improve several inflammatory disorders through regulation of eicosanoid production. However, since GLA is a precursor of arachidonic acid, it may bring about increasing tissue arachidonic acid levels with subsequent pro-inflammatory events. To explore this possibility, we examined the effect of high-dose GLA acid on the fatty acid profile of immune cells, leukotriene B4 production by peritoneal exudate cells and immunoglobulin productivity of mesenteric lymph node lymphocytes of Sprague-Dawley rats. Male rats were fed 10% fat diets containing graded levels, 0, 20, 40 and 60% of GLA for 3 weeks. The results showed the distinction in activity of metabolizing GLA between immune cells and liver. Thus, in immune cells such as mesenteric lymph node and spleen lymphocytes and peritoneal exudate cells, more dihomo-gamma-linolenic acid was found than in the liver. Leukotriene B4 production by peritoneal exudate cells was significantly suppressed when fed the highest level of GLA suggesting a lower risk of allergic reaction. Moreover, immunoglobulin productivity in mesenteric lymph node lymphocytes was promoted by dietary GLA. The present study indicates that a high dose of GLA may exert anti-inflammatory effects through suppression of leukotriene B4 release and strengthening of gut immune system, thus ameliorating allergic reaction.     

Mouse cytomegalovirus M33 is necessary and sufficient in virus-induced vascular smooth muscle cell migration

Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration.