Depletion of Blood-Borne Macrophages Does Not Reduce Demyelination in Mice Infected with a Neurotropic Coronavirus

Mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating disease with symptoms of hindlimb paralysis. Histological examination of the brains and spinal cords of these animals reveals the presence of large numbers of activated macrophages/microglia. In two other experimental models of demyelination, experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus-induced demyelination, depletion of hematogenous macrophages abrogates the demyelinating process. In both of these diseases, early events in the demyelinating process are inhibited by macrophage depletion. From these studies, it was not possible to determine whether infiltrating macrophages were required for late steps in the process, such as myelin removal. In this study, we show that when macrophages are depleted with either unmodified or mannosylated liposomes encapsulating dichloromethylene diphosphate, the amount of demyelination detected in MHV-infected mice is not affected. At a time when these cells were completely depleted from the liver, approximately equivalent numbers of macrophages were present in the spinal cords of control and drug-treated animals. These results suggest that blood-borne macrophages are not required for MHV-induced demyelination and also suggest that other cells, such as perivascular macrophages or microglia, perform the function of these cells in the presence of drug.     

High-Dose Borna Disease Virus Infection Induces a Nucleoprotein-Specific Cytotoxic T-Lymphocyte Response and Prevention of Immunopathology

Experimental Borna disease virus (BDV) infection of rats and natural infection of horses and sheep leads to severe central nervous system disease based on immunopathological pathways. The virus replicates slowly, and the cellular immune response results in immunopathology. CD8(+) T cells exert effector cell functions, and their activity results in the destruction of virus-infected cells. Previously, Oldach and colleagues (D. Oldach, M. C. Zink, J. M. Pyper, S. Herzog, R. Rott, O. Narayan, and J. E. Clements, Virology 206:426-434, 1995) have reported protection against Borna disease after inoculation of high-dose cell-adapted BDV. Here we show that the outcome of the infection, i.e., immunopathology versus protection, is simply dependent on the amount of virus used for infection. High-dose BDV (10(6) FFU) triggers an early virus-specific reaction of the immune system, as demonstrated by strong cellular and humoral responses. In particular, the early presence and function of nucleoprotein-specific CD8(+) T cells could be demonstrated in the brain. We present evidence that in a noncytolytic and usually persistent virus infection, high-dose input virus mediates early control of the pathogen due to an efficient induction of an antiviral immune mechanism. From these data, we conclude that immune reactivity, in particular the cytotoxic T-cell response, determines whether the virus is controlled with prevention of the ensuing immunopathological disease or whether a persistent infection is established.     

Cytotoxic T-Lymphocyte Precursor Frequencies in BALB/c Mice after Acute Respiratory Syncytial Virus (RSV) Infection or Immunization with a Formalin-Inactivated RSV Vaccine

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.Respiratory syncytial virus (RSV) infection in infants and young children often results in lower respiratory tract disease and is a high priority for vaccine development (1, 2). Attempts to develop an effective live, inactivated, or subunit vaccine have been unsuccessful (24, 25, 28). Early efforts at vaccinating young children with a formalin-inactivated RSV (FI-RSV) vaccine failed to protect the children from naturally acquired infection and actually enhanced lower respiratory tract disease upon later virus infection (2, 15, 24, 25). This enhanced disease has created concern about the safety of any nonlive RSV vaccine and, consequently, understanding the pathogenesis of FI-RSV-induced enhanced disease is critically important to vaccine development. Studies with BALB/c mice suggest that induction of memory T cells producing Th2-like cytokines, as a result of FI-RSV vaccination, may be key to the pathogenesis of enhanced disease (6, 16, 28, 32, 40). Th2-like cytokine mRNA has been demonstrated in cells from lung tissue or bronchoalveolar lavage (BAL) specimens after RSV challenge of FI-RSV-immunized mice (17, 32, 40). In addition, in vivo studies using antibody (Ab) blockade showed that the enhanced histopathology in FI-RSV-immune mice challenged with live virus could be eliminated by using anti-interleukin-4 (IL-4) and anti-IL-10 Abs but not anti-IL-12 Abs (6). Recent evidence suggests that CD8⁺ T lymphocytes may be important in directing the type of inflammatory response to RSV in challenge of G glycoprotein-sensitized mice (21, 31).One aspect of the FI-RSV immune response that has not been well characterized is the cytotoxic T-lymphocyte (CTL) response. There is limited information on major histocompatibility complex (MHC) class I-restricted CTLs after FI-RSV immunization (29), while the information about the CTL response after live-RSV infection has been well documented. Several studies have shown class I-restricted CTLs to kill predominantly target cells expressing the M, N, or F RSV protein (5, 7, 9, 26, 29, 41). The role of CTLs in the immune response to RSV is well illustrated by in vivo depletion studies with BALB/c mice (8, 18, 30). These studies suggest that both CD4⁺ (class II) and CD8⁺ lymphocytes are important for clearing RSV and that both contribute to the inflammatory response associated with infection. A vaccinia virus construct expressing RSV membrane-associated, nonglycosylated protein M2 has been affiliated with short-term protection in the BALB/c mouse (7). This protein does not induce neutralizing Abs, and therefore, protection likely is mediated by CTLs. Passive transfer of CD8⁺ T lymphocytes has been associated with both clearance of the virus and enhanced histopathology (1).In this report, we describe studies of CTL precursor (CTLp) frequencies in both live-RSV-infected and FI-RSV-immunized mice for MHC class I- and class II-restricted target cells. These studies demonstrate clear differences in the CTLp response between RSV and FI-RSV immunizations and provide additional approaches to identifying potential FI-RSV-induced enhanced disease mechanisms.     

Macrophage Infiltration, but Not Apoptosis, Is Correlated with Immune-Mediated Demyelination following Murine Infection with a Neurotropic Coronavirus

Mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis that is in large part immune mediated. Potential mechanisms of immune activity were assessed using an adoptive transfer system. Mice deficient in recombinase-activating gene function (RAG1(-/-)), defective in B- and T-cell maturation, become persistently infected with MHV but do not develop demyelination. Adoptive transfer of splenocytes from mice immunized to MHV into RAG1(-/-) mice infected with an attenuated strain of the virus results in the rapid and progressive development of demyelination. Most striking, adoptive transfer resulted, within 5 to 6 days, in extensive recruitment of activated macrophages/microglia to sites of demyelination within the spinal cord. Clearance of virus antigen occurred preferentially from the gray matter of the spinal cord. Apoptotic cells were identified in both the gray and white matter of the central nervous system (CNS) from RAG1(-/-) mice before and after adoptive transfer, with a moderate increase in number, but not distribution, of apoptotic cells following the development of demyelination. These results suggest that apoptosis following MHV-JHM infection of the murine CNS is not sufficient to cause demyelination. These results, showing that macrophage recruitment and myelin destruction occur rapidly after immune reconstitution of RAG(-/-) mice, suggest that this will be a useful system for investigating MHV-induced demyelination.     

Association between Virus-Specific Cytotoxic T-Lymphocyte and Helper Responses in Human Immunodeficiency Virus Type 1 Infection

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.     

Combination Therapy Using Chimeric Monoclonal Antibodies Protects Mice from Lethal H5N1 Infection and Prevents Formation of Escape Mutants

Background

Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants.

Methods/Principal Findings

We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy.

Conclusions/Significance

Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.     

Fetal Growth Retardation and Lack of Hypotaurine in Ezrin Knockout Mice

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez^−/−) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis–time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez^−/− mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.     

The Detection of Mutants in Mice by Electrophoresis: Results of a Model Induction Experiment with Procarbazine

Male mice of the DBA/2J strain were treated with the mutagen procarbazine and mated with C57BL/6J females. Offspring and parents were then analyzed for electrophoretically expressed mutations. A control group, not mutagen treated, was also examined. Two mutants probably due to induction were identified, and several of spontaneous origin were also found.     

Relation of Infection to Tissue Temperature in Mice Infected with Mycobacterium marinum and Mycobacterium leprae

Intravenous and footpad infections with Mycobacterium marinum and footpad infections with M. leprae were compared in the following mouse strains: A/He, BALB/C, CBA, C3H, C57BL, C57L, DBA, 101, and CFW. The results varied a great deal according to mouse strain used. Intravenous injection of high doses of M. marinum resulted in deaths after 28 days of 100% of strain A/He, and none of strain 101; 27 days after injection, the feet and noses of all strain CBA mice, but few of the C57BL, 101, or CFW mice, were involved. Injection of a small dose of M. marinum into the footpad produced visible disease in 5 days in all of the C57BL and 101 mice, but in not more than 60% of the A/He, DBA, and CFW mice; the average amount of swelling at 17 days varied from 4.40 mm in strain C57L to 0.92 in strain 101. After footpad injection of M. leprae, the average plateau harvests varied from 1.3 x 10(7) acid-fast bacteria in strain CBA to 6.5 x 10(5) in strain C57L. The infections in CBA mice extended from the site of inoculation throughout the foot. The temperature was measured rectally, in the footpad, and in the tail. Analysis of all the results revealed little correlation among the three types of infection. There was a strong negative correlation between the tail temperature and the death rate after intravenous injection of M. marinum, and a strong positive correlation between footpad temperature and plateau harvest of M. leprae.     

Strong Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T-Lymphocyte Activity in Sydney Blood Bank Cohort Patients Infected with nef-Defective HIV Type 1

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with natural nef and nef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.     

Ifit2 Deficiency Results in Uncontrolled Neurotropic Coronavirus Replication and Enhanced Encephalitis via Impaired Alpha/Beta Interferon Induction in Macrophages

Type I interferons (IFN-α/β) limit viral dissemination prior to the emergence of adaptive immune responses through the concerted action of interferon-stimulated genes (ISGs). Although IFN-α/β induction by coronaviruses is modest, it effectively limits viral spread within the central nervous system (CNS) and protects against mortality. The protective roles of specific ISGs against the mouse hepatitis virus (MHV) members of the coronaviruses are largely unknown. This study demonstrates a protective role of the ISG Ifit2 in encephalitis induced by the dual hepato- and neurotropic MHV-A59. Contrasting the mild encephalitis and 100% survival of MHV-A59-infected wild-type (wt) mice, nearly 60% of infected Ifit2^−/− mice exhibited severe encephalitis and succumbed between 6 and 8 days postinfection. Increased clinical disease in Ifit2^−/− mice coincided with higher viral loads and enhanced viral spread throughout the CNS parenchyma. Ifit2^−/− mice also expressed significantly reduced IFN-α/β and downstream ISG mRNAs Ifit1, Isg15, and Pkr, while expression of proinflammatory cytokines and chemokines was only modestly affected in the CNS. Impaired IFN-α/β induction in the absence of Ifit2 was confirmed by ex vivo mRNA analysis of microglia and macrophages, the prominent cell types producing IFN-α/β following MHV CNS infection. Furthermore, both IFN-α/β mRNA and protein production were significantly reduced in MHV-infected Ifit2^−/− relative to wt bone marrow-derived macrophages. Collectively, the data implicate Ifit2 as a positive regulator of IFN-α/β expression, rather than direct antiviral mediator, during MHV-induced encephalitis.     

Bortezomib Is Cytotoxic to the Human Growth Plate and Permanently Impairs Bone Growth in Young Mice

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.     

CD4 Depletion in SIV-Infected Macaques Results in Macrophage and Microglia Infection with Rapid Turnover of Infected Cells

In rhesus macaques (RMs), experimental depletion of CD4⁺ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4⁺ T-cells prior to SIVmac₂₅₁ infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA⁺ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4⁺ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.     

Mice Chronically Fed High-Fat Diet Have Increased Mortality and Disturbed Immune Response in Sepsis

Background

Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed.

Methodology/Principal Findings

The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5–7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1β. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice.

Conclusions

Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis.     

Oxidative Stress in Lungs of Mice Infected with Influenza a Virus

As oxidative stress has been implicated in the pathogenesis of certain viral diseases we determined antioxidant and prooxidant parameters in lungs and bronchoalveolar lavage fluid (BALF) of mice infected with a lethal dose of influenza A/PR8/34 virus. Viral infection was characterized by massive infiltration of leukocytes, mainly polymorphonuclear leukocytes, into the alveolar space. The total number of BALF cells increased up to 8-fold (day 3 post-infection) and these cells appeared activated as judged by their increased rates of superoxide anion radical (O₂^-) generation upon stimulation. Maximal rates of radical generation by BALF cells during the early stages of infection were 15- or 70-fold higher than those of cells from control animals when expressed per cell or total BALF cells, respectively. At the terminal stages of infection the total capacity of BALF cells to release of declined to ≈ 35-fold the control values. Infection also resulted in increased in vivo formation of hydrogen peroxide (H²O²) within the lungs at a time that coincided with the maximal capacity of BALF cells to release O_2-.

Whereas pulmonary activities of glutathione peroxidase and reductase remained unaltered, levels of ascorbate in the cell-free BALF decreased significantly during the early stages of the infection and then returned to normal levels and above, late in infection. The oxidation state of the dehydroascorbic acid/ ascorbate couple increased concomitantly with the decrease in ascorbate concentrations early in infection and remained elevated throughout the infection. As assessed by the prevention of peroxyl radical-induced loss of phycoerythrin fluorescence, the total antioxidant capacity present in lung tissue homogenate from terminally ill animals was not diminished when compared to that prepared from lungs of control mice. We conclude that although early stages of influenza infection are associated with the presence of oxidative stress in the lung tissue and alveolar fluid lining the epithelial cells, this stress does not appear to overwhelm local antioxidant defenses. The results therefore do not support a direct causative role of oxidative tissue damage in the pathogenesis of influenza virus infection.