Immune mechanisms in murine gammaherpesvirus-68 infection

The murine gamma-herpesvirus-68 (MHV-68) is a relative of the Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) that infects mice. All these gamma-herpesviruses are subject to immune control, but limit the impact of this control through immune evasion. Molecular evasion mechanisms have been described in abundance. However, we can only speculate what EBV and KSHV immune evasion contributes to the viral lifecycle. With MHV-68, we can analyze in vivo the contribution of immunological and virological gene expression to pathogenesis. While the physiology of infection seems quite well conserved between these viruses, the pathologies associated with immune suppression are obviously very different. MHV-68 is therefore more suited to uncovering the basic biology of gamma-herpesvirus infection than to testing disease interventions. Nevertheless, it may make some useful predictions about effective strategies of vaccination and infection control. This review aims to outline our current state of knowledge and to highlight some limitations of the MHV-68 model as it stands, in the hope of stimulating constructive progress.     

Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact

Abstract

Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.     

Murine gammaherpesvirus-68-induced interleukin-10 increases viral burden, but limits virus-induced splenomegaly and leukocytosis

Based on its genomic sequence and its pathogenesis, murine gammaherpesvirus-68 (gammaHV-68) has been established as a tractable model for the study of viral infections caused by the human gammaherpesviruses, Epstein-Barr virus or human herpesvirus-8. Despite significant advances, the mechanisms responsible for gammaHV-68-induced alterations in the protective host response, and the accompanying virus-induced leukocytosis, are not clear. In the present study, we questioned whether viral infection resulted in endogenous interleukin-10 (IL-10) production that might alter the host response. Infection of C57BL/6 mice resulted in increased IL-10 expression, demonstrating that gammaHV-68 could induce endogenous production of this cytokine. Infected C57BL/6 mice demonstrated the characteristic splenomegaly associated with this viral infection, however, we were surprised to discover that the splenomegaly was greater in syngeneic mice genetically deficient in IL-10 (IL-10-/-). These results strongly suggested that endogenously produced IL-10 might serve to limit leukocytosis in wild-type mice. Quantification of viral burden demonstrated a significant elevation in C57BL/6 versus IL-10-/- mice, with increases in virus being observed in both the macrophage and B-lymphocyte populations. The decreased viral load in syngeneic IL-10-/- mice correlated with an increased expression of endogenous IL-12, suggesting a mechanism of protection that was IL-12 dependent. Taken together, these studies demonstrate a surprising dichotomy for endogenous IL-10 production during gammaHV-68 infection. While the lack of IL-10 results in increased IL-12 expression and a lower viral burden, IL-10-/- mice also experience an increased leukocytosis.     

Murine IL-2 receptor beta chain blockade improves human leukocyte engraftment in SCID mice

Severe combined immunodeficient (SCID) mice accept human xenografts and can act as a model for human immune functions. Murine natural killer cells (NK), however, represent an important barrier for the reconstitution of SCID mice with human peripheral blood leukocytes (Hu-PBL). We investigated the effect on Hu-PBL survival of pretreatment with TM-β1, a rat monoclonal antibody for the mouse IL-2 receptor beta chain. TM-β1 greatly improved the survival of Hu-PBL. Human lymphocytes, predominantly T cells, survived in the peritoneum and infiltrated spleen and lungs already 1 week after engraftment and liver and thymus from 2 weeks on. Secondary humoral responses were evaluated with Hu-PBL from a donor immune to hepatitis-B surface Ag (HBsAg) and tetanus toxoid (TT). TM-β1 pretreatment enhanced the recall Ig response to HBsAg and did not affect the baseline anti-TT Ig production. In conclusion, TM-β1 pretreatment of SCID mice significantly improves the survival and functionality of the Hu-PBL graft.     

Adrenal chromaffin cells exhibit impaired granule trafficking in NCAM knockout mice

Neural cell adhesion molecule (NCAM) plays several critical roles in neuron path-finding and intercellular communication during development. In the clinical setting, serum NCAM levels are altered in both schizophrenic and autistic patients. NCAM knockout mice have been shown to exhibit deficits in neuronal functions including impaired hippocampal long term potentiation and motor coordination. Recent studies in NCAM null mice have indicated that synaptic vesicle trafficking and active zone targeting are impaired, resulting in periodic synaptic transmission failure under repetitive physiological stimulation. In this study, we tested whether NCAM plays a role in vesicle trafficking that is limited to the neuromuscular junction or whether it may also play a more general role in transmitter release from other cell systems. We tested catecholamine release from neuroendocrine chromaffin cells in the mouse adrenal tissue slice preparation. We utilize electrophysiological and electrochemical measures to assay granule recruitment and targeting in wild-type and NCAM -/- mice. Our data show that NCAM -/- mice exhibit deficits in normal granule trafficking between the readily releasable pool and the highly release-competent immediately releasable pool. This defect results in a decreased rate of granule fusion and thus catecholamine release under physiological stimulation. Our data indicate that NCAM plays a basic role in the transmitter release mechanism in neuroendocrine cells through mediation of granule recruitment and is not limited to the neuromuscular junction and central synapse active zones.     

Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and Toll-like receptor 2 knockout mice and protects against reinfection

Coxiella burnetii is a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuated C. burnetii phase II (having a truncated “O” chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-γ^−/−), BALB/c, Toll-like receptor 2 knockout (TLR2^−/−), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-γ^−/− and TLR2^−/− mice, which showed elevated rectal temperatures that were sustained for ~15 days with transient increases in splenic weights. Reinfection of IFN-γ^−/− and TLR2^−/− mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2^−/− mouse spleens was observed, greater colonization was evident in the IFN-γ^−/− mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-γ^−/− macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2^−/− macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.     

Cytokines and costimulatory molecules in the immune response to murine gammaherpesvirus-68

Murine gammaherpesvirus 68 (MHV-68) infection of mice provides a useful small animal model for studying gammaherpesvirus pathogenesis and immunity. Recent work has elucidated the cytokine and chemokine profiles during MHV-68 infection and has identified some of the costimulatory interactions that are important for an effective immune response to this virus. Several themes emerge from this work. There is a differential requirement for certain cytokines and costimulatory molecules in the acute and long-term control of MHV-68, and for control of the virus in different anatomical sites. CD4 T cell help is not required for short-term control of MHV-68 in the lung by cytotoxic CD8 T cells, but is essential for effective long-term control. Stimulation via CD40 is an important component of this CD4 T cell help, and interestingly, some of its effects appear to be independent of CD28. MHV-68 infection also increases the expression of several chemokines, which could potentially play important roles in leukocyte trafficking to sites of infection. However, to counter this response, MHV-68 has evolved strategies that enable it to evade or subvert the host chemokine system. Studying the role of cytokines and costimulatory molecules in immunity to MHV-68 may provide useful insights for the development of agents to control gammaherpesviruses that cause human disease.     

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.     

Meal patterns and foraging in melanocortin receptor knockout mice

We report the meal patterns of mice with the deletion of either the melanocortin type 3 or 4 receptors (MC3RKO or MC4RKO) compared with that of the wild type (WT) under conditions of varying foraging costs. Mice lived in two-lever operant chambers; the completion of a designated number of responses (termed procurement fixed ratio or PFR) on the "foraging" lever activated the other lever. On this second lever, the completion of a designated number of responses (termed consumatory fixed ratio or CFR) caused the delivery of a 20-mg food pellet. Animals could complete as many CFRs as they wished to constitute a meal, but whenever 10 min elapsed without pressing on this second lever, the meal was terminated and pressing on the "foraging" lever was again required to initiate a new meal. At lower PFRs, mice of all three genotypes took 5-7 well-defined meals per day of approximately 35 pellets/meal. At the highest PFR, mice of all three groups took about half this number of meals, with some increase in meal size, and total intake was slightly reduced. MC4RKO mice were obese compared with WT or MC3RKO but failed to eat more food in the operant chambers and, as a consequence, lost weight, regardless of PFR. Thus, changes in meal-taking strategies as a function of imposed foraging cost are not critically dependent on either MC3 or MC4 receptors, but these conditions did not allow us to study meal patterns in MC4RKO mice that are hyperphagic.     

雌激素受体α基因敲除对小鼠认知功能的影响机制

目的探讨ERα调节小鼠认知功能的作用及可能机制。方法将6只8～12周龄ERa基因敲除鼠和6只同周龄野生型小鼠分为2组：正常对照组和ERα基因敲除组。用水迷宫检测这2组小鼠的认知功能;ELISA法检测脑内Aβ含量;免疫组化法检测脑内Aβ沉淀情况。结果定位航行实验显示：与正常对照小鼠相比,ERα基因敲除鼠的逃避潜伏期显著延长（P〈O．01）;空间探索实验显示：ERα基因敲除鼠在目标象限的游泳距离占总的游泳距离的比例低于正常对照小鼠（P〈0．01）ELISA和免疫组化显示：与正常对照小鼠相比,ERα基因敲除鼠脑内Aβ生成增加（P〈0．01）。结论ERα基因敲除可导致认知功能下降,其机制可能与脑内Aβ生成增多有关。ERα基因敲除小鼠表现出类似阿尔芡海默病（AD）的病理以及行为学特征,提示其可以作为一种研究AD及其干预措施的新的动物模型。     

Murine polymorphonuclear neutrophils produce interferon-gamma in response to pulmonary infection with Nocardia asteroides

Nocardia asteroides causes an acute, necrotizing pneumonia characterized by extensive infiltration of polymorphonuclear neutrophils (PMNs) into the lungs. Although PMNs have historically been classified as end-point cells, recent investigations have indicated that PMNs have the ability to secrete cytokines such as interleukin (IL)-4 and IL-12. This study investigated the ability of PMNs to produce cytokines in a murine model of N. asteroides pulmonary infection. Flow cytometric analysis demonstrated the production of interferon-gamma (IFN-gamma), but not IL-4, by PMNs in response to this infection. IFN-gamma production correlated with peak infiltration of PMNs into the lungs. Cell sorting and enzyme-linked immunosorbent assay were used to confirm cytokine production by cells with nuclear morphology characteristic of PMNs. This is the first report of IFN-gamma production by neutrophils in response to an infection in vivo. These results suggest that PMNs play an important role in directing the host toward a T helper cell type 1 phenotypic response in the lungs.     

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice

The cystic fibrosis (Cftr and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expression in vivo, suggesting that the regulation of their expression may be co- ordinated. We have tested this hypothesis in vivo by examining Cftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftr(tm1CAM)). Cftr mRNA expression in Cftr(tm1CAM)/Cftr(tm1CAM) mice was 4-fold reduced in the intestine, as compared with littermate wild- type mice. All other Cftr(tm1CAM)/Cftr(tm1CAM) mouse tissues examined showed similar reductions in Cftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-old Cftr(tm1CAM)/Cftr(tm1CAM) mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygous Cftr(tm1CAM) mice. In 10-week-old, Cftr(tm1CAM)/Cftr(tm1CAM) mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes, Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in the Cftr(tm1CAM)/Cftr(tm1CAM) mice are specific to the loss of Cftr expression and/or function. These data provide further evidence supporting the hypothesis that the regulation Cftr and Mdr1 expression is co-ordinated in vivo, and that this co-ordinate regulation is influenced by temporal factors.      

Multiple histamine receptor gene knockout mice and their phenotypes

Inflammation Research -     

Baroreflex sensitivity and oxidative stress in the LDL receptor knockout mice

This study aims at observing the effect of low-density lipoprotein (LDL) receptor deficiency in cholesterol blood levels, baroreflex sensitivity (BRS), nitric oxide (NO) bioavailability, and oxidative stress. The lack of LDL receptors in mice significantly increased the cholesterol blood levels (179+/-35 vs. 109+/-13mg/dL) in the knockout (KO) mice compared to control. There was no difference in basal mean arterial pressure and heart rate between the groups. However, in KO mice the BRS was significantly attenuated and the antioxidant enzyme activities, measured in erythrocytes and heart, were significantly decreased. On the other hand, the oxidative damage measured by chemiluminescence and carbonyls was increased, while total plasma nitrate levels were lower in KO mice, indicating a decrease in NO availability. In conclusion, these results indicate that the lack of LDL receptor increased cholesterol blood levels, induced oxidative stress and decreased BRS.     

Murine scrapie infection causes an abnormal germinal centre reaction in the spleen

Follicular dendritic cells (FDCs) of the lymphoreticular system play a role in the peripheral replication of prion proteins in some transmissible spongiform encephalopathies (TSEs), including experimental murine scrapie models. Disease-specific PrP (PrPd) accumulation occurs in association with the plasmalemma and extracellular space around FDC dendrites, but no specific immunological response has yet been reported in animals affected by TSEs. In the present study, morphology (light microscopical and ultrastructural) of secondary lymphoid follicles of the spleen were examined in mice infected with the ME7 strain of scrapie and in uninfected control mice, with or without immunological stimulation with sheep red blood cells (SRBCs), at 70 days post-inoculation or at the terminal stage of disease (268 days). Scrapie infection was associated with hypertrophy of FDC dendrites, increased retention of electron-dense material at the FDC plasma membrane, and increased maturation and numbers of B lymphocytes within secondary follicles. FDC hypertrophy was particularly conspicuous in immune-stimulated ME7-infected mice. The electron-dense material was associated with PrP Napoli accumulation, as determined by immunogold labelling. We hypothesize that immune system changes are associated with increased immune complex trapping by hypertrophic FDCs expressing PrP Napoli molecules at the plasmalemma of dendrites, and that this process is exaggerated by immune system stimulation. Contrary to previous dogma, these results show that a pathological response within the immune system follows scrapie infection.     

Effects of interferon-gamma treatment on surgically simulated wound infection in mice

Interferon-gamma (IFN-gamma) has been shown to have immunoregulatory properties and is able to modulate resistance to several microbial infections. This study was designed to determine the efficacy of IFN-gamma treatment in a mouse model of infection that simulates many clinical conditions associated with surgical wound infection: viz, a bacteria laden suture and tissue injury. The test bacteria were Klebsiella pneumoniae. Groups of CBA/J mice received either IFN-gamma or RPMI-1640 medium (controls) subcutaneously. IFN-gamma was administered daily at a dose of 7500 units for 5 days prior to bacterial challenge. Mice treated with IFN-gamma survived significantly longer than controls. Systemic bacterial recovery was significantly reduced in the IFN-gamma treated group but local bacterial recovery was unaffected.     

Murine models of acute and chronic lung infection with cystic fibrosis pathogens

Animal models of acute and chronic infection, along with mice genetically modified for the Cftr gene, are a key asset in cystic fibrosis (CF) research. Despite some limitations, these models provide valuable resources to mimic the initial and progressive bronchopulmonary infection typical of CF patients. The following review summarizes the strengths and weaknesses of different types of animal models with a major emphasis placed on the significant species differences between mice and humans. Murine models of acute and chronic lung infection with Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, and Haemophilus influenzae have been used to study the molecular mechanisms underlying the pathogen virulence and host defense. In addition, they have provided insights in the potential of vaccination to restrict infectious exacerbations, the activity of antibiotics, and the effectiveness of anti-inflammatory therapy in reducing lung damage. Indeed, animal models of infection should allow the validation of future therapeutic interventions for lung infections in patients with CF.     

Chemokines and leukocyte trafficking in rheumatoid arthritis.

Leukocyte infiltration into the joint space and tissues is an essential component of the pathogenesis of rheumatoid arthritis (RA). In this review, we will summarize the current understanding of the mechanisms of leukocyte trafficking into the synovium, focusing on the role of adhesion molecules, chemokines, and chemokine receptors in synovial autoimmune inflammation. The process by which a circulating leukocyte decides to migrate into the synovium is highly regulated and involves the capture, firm adhesion, and transmigration of cells across the endothelial monolayer. Adhesion molecules and chemokine signals function in concert to mediate this process and to organize leukocytes into distinct structures within the synovium. Chemokines play a key regulatory role in organ-specific leukocyte trafficking and activation by affecting integrin activation, chemotaxis, effector cell function, and cell survival. Consequently, chemokines, their receptors, and downstream signal transduction molecules are attractive therapeutic targets for RA.