Control of Early Theiler's Murine Encephalomyelitis Virus Replication in Macrophages by Interleukin-6 Occurs in Conjunction with STAT1 Activation and Nitric Oxide Production

During Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages, it is thought that high interleukin-6 (IL-6) levels contribute to the demyelinating disease found in chronically infected SJL/J mice but absent in B10.S mice capable of clearing the infection. Therefore, IL-6 expression was measured in TMEV-susceptible SJL/J and TMEV-resistant B10.S macrophages during their infection with TMEV DA strain or responses to lipopolysaccharide (LPS) or poly(I · C). Unexpectedly, IL-6 production was greater in B10.S macrophages than SJL/J macrophages during the first 24 h after stimulation with TMEV, LPS, or poly(I · C). Further experiments showed that in B10.S, SJL/J, and RAW264.7 macrophage cells, IL-6 expression was dependent on extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) and enhanced by exogenous IL-12. In SJL/J and RAW264.7 macrophages, exogenous IL-6 resulted in decreased TMEV replication, earlier activation of STAT1 and STAT3, production of nitric oxide, and earlier upregulation of several antiviral genes downstream of STAT1. However, neither inhibition of IL-6-induced nitric oxide nor knockdown of STAT1 diminished the early antiviral effect of exogenous IL-6. In addition, neutralization of endogenous IL-6 from SJL/J macrophages with Fab antibodies did not exacerbate early TMEV infection. Therefore, endogenous IL-6 expression after TMEV infection is dependent on ERK MAPK, enhanced by IL-12, but too slow to decrease viral replication during early infection. In contrast, exogenous IL-6 enhances macrophage control of TMEV infection through preemptive antiviral nitric oxide production and antiviral STAT1 activation. These results indicate that immediate-early production of IL-6 could protect macrophages from TMEV infection.     

Role of Interleukin-2 and Herpes Simplex Virus 1 in Central Nervous System Demyelination in Mice

We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.     

High Numbers of Viral RNA Copies in the Central Nervous System of Mice during Persistent Infection with Theiler's Virus

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 x 10(9)) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.     

Different Subcellular Localization of Theiler's Murine Encephalomyelitis Virus Leader Proteins of GDVII and DA Strains in BHK-21 Cells

The highly virulent GDVII strain of Theiler''s murine encephalomyelitis virus causes acute and fatal encephalomyelitis, whereas the DA strain causes mild encephalomyelitis followed by a chronic inflammatory demyelinating disease with virus persistence. The differences in the amino acid sequences of the leader protein (L) of the DA and GDVII strains are greater than those for any other viral protein. We examined the subcellular distribution of DA L and GDVII L tagged with the FLAG epitope in BHK-21 cells. Wild-type GDVII L was localized predominantly in the cytoplasm, whereas wild-type DA L showed a nucleocytoplasmic distribution. A series of the L mutant experiments demonstrated that the zinc finger domain, acidic domain, and C-terminal region of L were necessary for the nuclear accumulation of DA L. A GDVII L mutant with a deletion of the serine/threonine (S/T)-rich domain showed a nucleocytoplasmic distribution, in contrast to the predominant cytoplasmic distribution of wild-type GDVII L. A chimeric DA/GDVII L, D/G, which encodes the N region of DA L including the zinc finger domain and acidic domain, followed by the GDVII L sequence including the S/T-rich domain, was distributed exclusively throughout the cytoplasm but not in the nucleus, as observed with wild-type GDVII L. Another chimeric L, G/D (which is the converse of the D/G construct), accumulated in the nucleus as well as the cytoplasm, as was observed for wild-type DA L. The findings suggest that the differential distribution of DA L and GDVII L is determined primarily by the S/T-rich domain. The S/T-rich domain may be important for the viral activity through the regulation of the subcellular distribution of L.Theiler''s murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus of the family Picornaviridae, and its strains are divided into two subgroups on the basis of their different biological activities. The neurovirulent strains, such as GDVII and FA, produce acute and fatal encephalomyelitis in mice. The persistent strains, such as TO, DA, BeAn, etc., induce mild and nonfatal encephalomyelitis, followed by a chronic demyelinating disease with virus persistence in the spinal cords of mice. This late demyelinating disease is thought to be an excellent experimental model for the human demyelinating disease multiple sclerosis (MS) (5, 17, 20).The TMEV genome is a single-stranded RNA molecule and translated as a long precursor polyprotein to yield 12 viral proteins by autoproteolytic cleavage (23). Two subgroup strains of TMEV have a sequence identity of approximately 95% at the amino acid level. The amino acid sequences of the proteins encoded by the P1, P2, and P3 regions of both strains are highly conserved and show 94, 96, and 98% identity, respectively. The genome has another coding region, designated the leader (L), at the most amino-terminal location of the precursor polyprotein. The L coding region encodes 76 amino acids (aa) and shows a low sequence identity of only 85% to the above-described three regions (16, 19, 22). Therefore, L has the greatest difference in amino acid sequence among any of the viral proteins and may play an important role in subgroup-specific biological activities of TMEV. In this study, we have investigated the subcellular localization of the L proteins of GDVII and DA strains and characterized the functional domains involved in the differential distribution between DA L and GDVII L in BHK-21 cells by a series of deletion mutant and chimeric construct experiments.     

Role of Murine Intestinal Interleukin-1 Receptor 1-Expressing Lymphoid Tissue Inducer-Like Cells in Salmonella Infection

Interleukin (IL)-1 signaling plays a critical role in intestinal immunology. Here, we report that the major population of intestinal lamina propria lymphocytes expressing IL-1 receptor 1 (IL-1R1) is the lymphoid tissue inducer (LTi)-like cell, a type of innate lymphoid cell. These cells are significant producers of IL-22, and this IL-22 production depends on IL-1R1 signaling. LTi-like cells are required for defense against Salmonella enterica serovar Typhimurium. Moreover, colonic LTi-like cell numbers depend on the presence of the intestinal microbiota. LTi-like cells require IL-1R1 for production of protective cytokines and confer protection in infectious colitis, and their cell numbers in the colon depend upon having a microbiome.     

Interleukin-6, a Major Cytokine in the Central Nervous System

Interleukin-6 (IL-6) is a cytokine originally identified almost 30 years ago as a B-cell differentiation factor, capable of inducing the maturation of B cells into antibody-producing cells. As with many other cytokines, it was soon realized that IL-6 was not a factor only involved in the immune response, but with many critical roles in major physiological systems including the nervous system. IL-6 is now known to participate in neurogenesis (influencing both neurons and glial cells), and in the response of mature neurons and glial cells in normal conditions and following a wide arrange of injury models. In many respects, IL-6 behaves in a neurotrophin-like fashion, and seemingly makes understandable why the cytokine family that it belongs to is known as neuropoietins. Its expression is affected in several of the main brain diseases, and animal models strongly suggest that IL-6 could have a role in the observed neuropathology and that therefore it is a clear target of strategic therapies.     

A Determinant for Central Nervous System Persistence Localized in the Capsid of Theiler’s Murine Encephalomyelitis Virus by Using Recombinant Viruses

The demyelinating process in Theiler’s murine encephalomyelitis virus (TMEV) infection in mice requires virus persistence in the central nervous system. Using recombinant TMEV assembled between the virulent GDVII and less virulent BeAn virus cDNAs, we now provide additional evidence supporting the localization of a persistence determinant to the leader P1 (capsid) sequences. Further, recombinant viruses in which BeAn sequences progressively replaced those of GDVII within the capsid starting at the leader NH2 terminus suggest that a conformational determinant requiring homologous sequences in both the VP2 puff and VP1 loop regions, which are in close contact on the virion surface, might underlie persistence.     

Priming of CD8+ T Cells during Central Nervous System Infection with a Murine Coronavirus Is Strain Dependent

Virus-specific CD8⁺ T cells are critical for protection against neurotropic coronaviruses; however, central nervous system (CNS) infection with the recombinant JHM (RJHM) strain of mouse hepatitis virus (MHV) elicits a weak CD8⁺ T-cell response in the brain and causes lethal encephalomyelitis. An adoptive transfer model was used to elucidate the kinetics of CD8⁺ T-cell priming during CNS infection with RJHM as well as with two MHV strains that induce a robust CD8⁺ T-cell response (RA59 and SJHM/RA59, a recombinant A59 virus expressing the JHM spike). While RA59 and SJHM/RA59 infections resulted in CD8⁺ T-cell priming within the first 2 days postinfection, RJHM infection did not lead to proliferation of naïve CD8⁺ T cells. While all three viruses replicated efficiently in the brain, only RA59 and SJHM/RA59 replicated to appreciable levels in the cervical lymph nodes (CLN), the site of T-cell priming during acute CNS infection. RJHM was unable to suppress the CD8⁺ T-cell response elicited by RA59 in mice simultaneously infected with both strains, suggesting that RJHM does not cause generalized immunosuppression. RJHM was also unable to elicit a secondary CD8⁺ T-cell response in the brain following peripheral immunization against a viral epitope. Notably, the weak CD8⁺ T-cell response elicited by RJHM was unique to CNS infection, since peripheral inoculation induced a robust CD8⁺ T-cell response in the spleen. These findings suggest that the failure of RJHM to prime a robust CD8⁺ T-cell response during CNS infection is likely due to its failure to replicate in the CLN.Members of the family Coronaviridae infect a wide range of mammalian species, including humans, and induce mild to severe disease of the respiratory tract, gastrointestinal tract, and central nervous system (CNS). Mouse hepatitis virus (MHV) infection provides a useful model for the study of acute and chronic CNS disease and specifically the process of demyelination, the hallmark of the human disease multiple sclerosis. Different strains of MHV induce disease with various degrees of severity. For example, CNS infection with the recombinant wild-type A59 (RA59) strain causes acute encephalitis during the first week of infection; a strong CD8⁺ T-cell response is observed in the brain, coinciding with viral clearance. However, despite clearance of infectious RA59 virus, demyelination develops, peaking at approximately 4 weeks postinfection (p.i.) (17, 20). In contrast, infection with the recombinant wild-type JHM (RJHM) strain (derived from the JHM isolate referred to as MHV-4 or JHM.SD [7, 28]) causes severe encephalomyelitis; the virus is not cleared, and mice typically succumb to disease by the end of the first week of infection. Furthermore, RJHM infection of the CNS elicits a very weak virus-specific CD8⁺ T-cell response in the brain (7, 20, 34). However, we have examined only the most virulent strain of JHM. It should be noted that there are other strains of JHM that have deletions and mutations within the spike glycoprotein, rendering them less virulent and sometimes resulting in a change in cell tropism. The ability of neurotropic strains of MHV to replicate within cells of the CNS and cause disease of various degrees is ideal for allowing the dissection of both viral and host determinants of neuropathogenesis.The spike glycoprotein of MHV is a major determinant of neurovirulence (32). It controls virus tropism and spread as it both binds the cellular receptor and induces fusion with target cells. In addition, it encodes neutralizing antibody epitopes and the H-2^b-restricted CD8⁺ T-cell epitopes recognized in C57BL/6 (B6) mice. The A59 spike differs from the JHM spike in that it contains a deletion of 52 amino acids within the hypervariable region. The hypervariable region has been well documented to tolerate mutation, but with attenuating effects on virulence (5, 7). RA59 and RJHM both encode an H-2K^b epitope at positions S598 to S605 (S598); however, due to the deletion, the A59 spike lacks the immunodominant H-2D^b epitope at positions S510 to S519 (S510). We previously selected isogenic recombinant viruses expressing the JHM spike in which all other genes are derived from the A59 strain of MHV (SJHM/RA59). The isogenic SJHM/RA59 virus has a 50% lethal dose (LD₅₀) similar to that of RJHM, demonstrating that the JHM spike is sufficient to generate a highly neurovirulent phenotype and an increased ability to spread within the CNS (32, 33). However, SJHM/RA59-infected mice exhibit slower kinetics of death than RJHM-infected mice, and notably, unlike RJHM, the chimeric SJHM/RA59 virus induces a strong CD8⁺ T-cell response in the brain (14, 34).In addition to the spike, there is increasing evidence that other viral genes play important roles in pathogenesis. We (14, 21) and others (34, 35) have noted that the low CD8⁺ T-cell response observed during RJHM infection is not dependent on the spike, since the SJHM/RA59 recombinant induces a robust virus-specific CD8⁺ T-cell response. The difference between the CD8⁺ T-cell responses elicited by SJHM/RA59 and RJHM may explain why SJHM/RA59 kills mice more slowly than RJHM. Furthermore, the reverse chimeric recombinant virus expressing the A59 spike in the JHM background (SA59/RJHM) is unable to replicate in the liver despite the fact that it expresses the spike from the hepatotropic RA59 strain (27), suggesting that background genes play a significant role in viral tropism.It is well established that virus-specific CD8⁺ T cells play a protective role against MHV and are essential for clearance of infectious virus from the CNS (6, 20, 40, 41). The effector mechanisms exerted by activated, virus-specific CD8⁺ T cells include the ability to secrete cytokines and the ability to lyse target cells. Gamma interferon (IFN-γ) expression is essential for clearance of MHV from the brain (3, 22, 29), and perforin-mediated lysis of infected cells also appears to play a role in viral clearance (6, 31). In contrast to infection with RA59 or the relatively neuroattenuated glial-cell-tropic strains of JHM, CNS infection with the highly neurovirulent RJHM strain results in very low levels of activated, virus-specific CD8⁺ T cells in the spleen and brain (14, 34). Furthermore, RJHM infection induces a different profile of cytokines and chemokines in the brains of infected mice than infection with RA59 (34, 35, 38). One dramatic difference is that RA59 infection results in a robust IFN-γ response whereas RJHM infection results in higher, sustained levels of IFN-β (34). These observations prompted us to address the following questions. (i) Does RJHM elicit a CD8⁺ T-cell response in the brain following intranasal (i.n.) inoculation, a route that requires more virus and results in slower infection than intracranial (i.c.) inoculation? (ii) What are the kinetics of CD8⁺ T-cell priming during CNS infections with RA59, SJHM/RA59, and RJHM? (iii) Is CNS infection with RJHM generally immunosuppressive? (iv) Do RA59, SJHM/RA59, and RJHM replicate efficiently in the draining cervical lymph nodes (CLN)? (v) Can RJHM elicit a secondary CD8⁺ T-cell response in the brain following peripheral immunization against a viral epitope? (vi) Is the low CD8⁺ T-cell response elicited during RJHM infection an inherent characteristic of the viral strain or specific to RJHM infection of the CNS? Our results suggest that RJHM fails to prime a CD8⁺ T-cell response specifically during infection of the CNS without causing generalized immunosuppression and that this lack of priming correlates with a low level of RJHM replication in the draining CLN, the site of CD8⁺ T-cell priming during acute CNS infection.     

Astrocyte-Derived CXCL10 Drives Accumulation of Antibody-Secreting Cells in the Central Nervous System during Viral Encephalomyelitis

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10^−/− but not CXCL9^−/− mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10^−/− mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9^−/− mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9^−/− or CXCL10^−/− mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.     

腺苷的中枢作用

腺苷是包括中枢神经系统（ＣＮＳ）细胞外液在内的体液的正常组成成分,其正常水平为０．０３～０．３μｍｏｌ／Ｌ。ＡＴＰ合成与分解失衡的条件下明显升高,如缺血时可升高１０００倍之多。腺苷通过腺苷受体（ａｄｅｎｏｄｉｎｅｒｅｃｅｐｔｏｒ,ＡＲ）对ＣＮＳ具有多方面的生理与病理作用,被认为是ＣＮＳ的抑制性神经调质,具有神经保护作用。     

Transgenic Expression of the 3D Polymerase Inhibits Theiler's Virus Infection and Demyelination

The RNA-dependent RNA polymerase 3D^pol is required for the elongation of positive- and negative-stranded picornavirus RNA. During the course of investigating the effect of the transgenic expression of viral genes on the host immune response, we evaluated the viral load present in the host after infection. To our surprise, we found that 3D transgenic expression in genetically susceptible FVB mice led to substantially lower viral loads after infection with Theiler''s murine encephalomyelitis virus (TMEV). As a result, spinal cord damage caused by chronic viral infection in the central nervous system was reduced in FVB mice that expressed 3D. This led to the preservation of large-diameter axons and motor function in these mice. The 3D transgene also lowered early viral loads when expressed in FVB-D^b mice resistant to persistent TMEV infection. The protective effect of 3D transgenic expression was not altered in FVB-Rag^−/−.3D mice that are deficient in T and B cells, thus ruling out a mechanism by which the overexpression of 3D enhanced the adaptive immune clearance of the virus. Understanding how endogenously overexpressed 3D polymerase inhibits viral replication may lead to new strategies for targeting therapies to all picornaviruses.Picornavirus infection is a major contributor to worldwide disease. Diseases such as poliomyelitis and hand-foot-and-mouth disease can be fatal. Other picornaviruses, such as rhinovirus, are partly responsible for upper respiratory tract infections. There are no drugs to treat picornavirus illness. However, some therapies show promise in vitro and in animal models. Winthrop compounds, which bind to hydrophobic sites on the surface of the virion that are important in viral attachment to the host and uncoating, decreased the number of upper respiratory symptoms following challenge with coxsackie virus 21 (45). A similar pocket binding drug, pleconaril (VP63843), showed 95% inhibition against 215 non-polio enteroviruses (39). A phase II trial of this drug against enteroviral meningitis decreased disease duration compared to the placebo (1). However, these drugs have side effects and were less effective in larger studies. Another limitation is that mutant viruses arose that sterically inhibited binding by substituting a bulky amino acid in the binding pocket (21). The administration of small interfering RNA (siRNA) has shown some promise in controlling picornavirus infections; however, the development of a delivery system is a major hurdle (8-10, 44).The picornavirus Theiler''s murine encephalomyelitis virus (TMEV) is a member of the Cardiovirus genus. TMEV is divided into two groups based on disease in mice after intracerebral injection (12, 15). The highly virulent GDVII subgroup causes fatal encephalitis, and the lowly virulent Theiler''s original (TO) subgroup, which includes BeAn and DA strains, causes a persistent infection in the white matter of the central nervous system (CNS), leading to chronic inflammation and demyelination in genetically susceptible mice. Chronic inflammation and demyelination leads to secondary axonal dysfunction and paralysis. Therefore, the BeAn and DA strains of TMEV infection in mice are used as animal models of demyelinating diseases such as multiple sclerosis (4, 11, 13).Inbred strains of mice differ in their susceptibility to TMEV (14, 18). Resistance to persistent infection depends on the haplotype of the major histocompatibility complex (H-2). Mice of the H-2^b,d,k haplotype are resistant to persistent infection, whereas mice of the H-2^f,p,q,r,s,v haplotype are susceptible to persistent infection (32). Resistance to persistent infection has been further defined to the D locus of H-2 (33, 35). The mice used in this paper all are on an FVB/NJ background. Due to the prominent pronuclei in their fertilized eggs and large litter size, FVB/NJ mice commonly are used for transgenic injection (43). These mice are of the H-2^q haplotype and are susceptible to persistent TMEV infection. Persistent infection leads to chronic spinal cord inflammation and demyelination in all inbred mice that are genetically susceptible to TMEV. FVB-D^b mice contain the H-2D^b transgene, which confers resistance to persistent TMEV infection (2). FVB mice and FVB-D^b mice have similar early acute disease in the brain at 7 days postinfection (dpi); however, unlike FVB mice, FVB-D^b mice control the virus in the brain and spinal cord by day 45 and do not develop demyelination.Picornaviruses perform multiple tasks inside host cells for successful viral replication, with very few gene products being responsible for these tasks. The single-stranded RNA picornavirus genome has, on average, 7,500 nucleotides and produces a single polyprotein that is cleaved by its own virus-encoded proteases. One of these proteins, the RNA-dependent RNA-polymerase 3D^pol, is required for the elongation of positive- and negative-stranded viral RNA. 3D^pol oligomerizes, which favors elongation and binding to RNA (16). 3D^pol forms a membranous replication complex with VPg and precursor proteins 3AB and 3CD to initiate VPg uridylylation, which serves as a primer for positive- and negative-strand RNA replication by 3D^pol (25, 40, 42). The stimulatory effect of 3AB on RNA replication by 3D^pol is inhibited by increasing concentrations of 3D (26, 31).During the course of investigating the effect of the transgenic expression of viral genes on the host immune response to TMEV, we evaluated the viral load present in the host after infection. Mice expressing the 3D transgene were used as control mice, since previous studies have shown that the 3D protein was ignored by T and B cells in the immune system (3, 22, 29). To our surprise, we found that the 3D transgenic mice substantially reduced TMEV in vivo. Therefore, we set out to study the effect of 3D overexpression in a transgenic mouse model of TMEV infection.