key: cord-0005769-yr45r6ky authors: Chen, Benjamin P.; Lane, Thomas E. title: Lack of nitric oxide synthase type 2 (NOS2) results in reduced neuronal apoptosis and mortality following mouse hepatitis virus infection of the central nervous system date: 2002 journal: J Neurovirol DOI: 10.1080/135502802317247820 sha: 3a6afafa2b93f6410bea960aeee72d00ab6269ba doc_id: 5769 cord_uid: yr45r6ky The role of nitric oxide synthase type-2 (NOS2)-derived nitric oxide (NO) in the pathogenesis of mouse hepatitis virus (MHV)-induced central nervous system disease was examined. Infection of NOS2 knockout ((−/−)) and NOS2(+/+) mice with MHV resulted in similar kinetics of viral clearance from the brain and comparable levels of demyelination. MHV-infected NOS2(−/−) mice displayed a marked decrease in mortality as compared to infected NOS2(+/+) mice that correlated with a significant decrease (P ≤ 0.001) in the number of apoptotic cells (determined by TUNEL staining) present in the brain. Confocal microscopy revealed that the majority of cells (>70%) undergoing apoptosis were neurons. These studies indicate that NOS2-generated NO contributes to apoptosis of neurons but not demyelination following MHV infection. Nitric oxide synthase-2 (NOS2) is the Ca 2Cindependent, inducible isoform of the NOS family with activity that results in high level expression of the free radical nitric oxide (NO) over an extended period of time (reviewed in MacMicking et al, 1997b) . NOS2 is expressed by a variety of cell types including macrophage/microglia and astrocytes following exposure to various signals, including in ammatory cytokines such as IFN-°and TNF-®. As such, numerous studies have determined that NOS2-generated NO is an important component in host defense against microbial infection (Adams et al, 1990; Karupiah et al, 1993; Lane et al, 1994; MacMicking et al, 1997a; Zaragoza et al, 1999) . In addition, NOS2-generated NO may contribute to various in ammatory pathologies by exerting a cytostatic and/or cytotoxic effect (Stuehr and Nathan, 1989; Campbell, 1996; Adler et al, 1997) . Infection of susceptible mice with MHV results in an acute encephalomyelitis followed by a chronic demyelinating disease with many clinical and histologic similarities to the human demyelinating disease multiple sclerosis (MS) (Houtman and Fleming, 1996; Lane and Buchmeier, 1997a) . NOS2 is expressed during both the acute and chronic disease stages of MHV infection suggesting a role for NOS2-derived NO in the pathogenesis of disease (Sun et al, 1995; Grzybicki et al, 1997; Lane et al, 1997b Lane et al, , 1999 . A recent study demonstrated that administration of aminoguanidine (AG), a relatively selective inhibitor of NOS2 activity, to MHV-infected mice resulted in reduced demyelination early following infection. However, demyelination was not affected at later time postinfection in AG-treated mice, suggesting a transient role for NO in myelin destruction (Lane et al, 1999) . The current study utilizes mice with a homozygous deletion in the nos2 gene (NOS2 ¡=¡ ) to further characterize the contributions of NOS2-generated NO in MHV-induced CNS disease. The studies presented demonstrate no signi cant differences in the severity of demyelination between MHV-infected NOS2 ¡=¡ mice and NOS2 C=C mice. However, MHV-infected NOS2 ¡=¡ mice displayed a marked decrease in mortality that correlated with a signi cant reduction ( P · 0.001) in numbers of apoptotic neurons within the CNS as compared to infected NOS2 C=C mice. Collectively, these data suggest that NOS2-generated NO contributes to acute neurologic disease by inducing apoptosis of neurons but does not have a role in demyelination. NOS2 C=C (5-6 weeks, C57BL/6, H-2 b background, Jackson Laboratories, Bar Harbor, Maine) and NOS2 ¡=¡ mice (5-6 weeks, C57BL/6-Nos2 tm1Lau , Jackson Laboratories) were infected intracranially (i.c.) with 10 plaque-forming units (PFU) of MHV strain V5A13.1 (Dalziel et al, 1986) . MHV infection of NOS2 C=C mice resulted in earlier onset and more severe clinical disease that correlated with a marked increase in mortality as compared to MHV-infected NOS2 ¡=¡ mice ( Figure 1 ). Differences in mortality Figure 1 Mortality in MHV-infected NOS2 C=C and NOS2 ¡=¡ mice. Five-to six-week-old NOS2 C=C and NOS2 ¡=¡ mice were infected intracranially with 10 PFU of MHV-V5A13.1. By day 12 p.i., greater than 85% of MHV-infected NOS2 ¡=¡ mice had died, whereas only 13% of the NOS2 ¡=¡ mice died. NOS2 C=C mice, nD 55; NOS2 ¡=¡ mice, nD 48. a All data are shown as mean § SEM. b Below level of detection of plaque assay (·100 PFU/g tissue). c Scoring was as follows: 0, no demyelination; 1, mild in ammation accompanied by loss of myelin integrity; 2, moderate in ammation with increasing myelin damage; 3, numerous in ammatory lesions accompanied by signi cant increase in myelin stripping; and 4, intense areas of in ammation accompanied by numerous phagocytic cells engul ng myelin debris . between the two populations of mice were notable beginning at day 7 p.i. and increased until day 12 p.i., at which point approximately 85% of MHV-infected NOS2 ¡=¡ mice survived infection as compared to less than 15% survival in the MHV-infected NOS2 C=C mice. The increase in disease severity in NOS2 C=C mice was not the result of higher viral titers or delayed clearance of virus from the CNS as infected NOS2 C=C and NOS2 ¡=¡ mice displayed comparable viral titers within the brains at all time points examined as assessed by plaque assay (Hirano et al, 1978 ) (Table 1). These data are consistent with previous studies that indicated that NOS2-generated NO does not contribute to clearance of MHV from the CNS (Lane et al, 1997b (Lane et al, , 1999 Wu et al, 2000) . To evaluate the severity of demyelination, spinal cords from MHVinfected NOS2 C=C and NOS2 ¡=¡ mice were removed at days 12 and 35 p.i. Examination of luxol fast blue-stained spinal cords indicated no signi cant differences in the severity of demyelination between NOS2 ¡=¡ and NOS2 C=C mice at either 12 or 35 days p.i., suggesting that NO does not play an important role contributing to MHV-induced demyelination (Table 1) . Previous studies have shown that NO is capable of triggering events leading to apoptosis (Mebmer et al, 1996; Melkova et al, 1997; Allione et al, 1999) . To determine if the diminished disease severity observed in MHV-infected NOS2 ¡=¡ mice was associated with a reduction in the number of cells undergoing apoptosis, TUNEL staining was performed by FACS analysis (FACStar, Beckton Dickinson, Mountain View, California) on cells obtained from the brains of MHVinfected mice by centrifugation through a discontinuous Percoll gradient . TUNEL analysis was performed using a uorescein death detection kit (Boehringer Mannheim, Indianapolis, Indiana). FACS analysis of TUNEL-positive cells present within the brains of infected mice revealed that NOS2 ¡=¡ mice displayed reduced levels of positive cells at all time points examined (days 3, 5, and 7 p.i.) as compared to infected NOS2 C=C mice (Figure 2 ). Examination of brains of MHV-infected NOS2 C=C and NOS2 ¡=¡ mice suggested that neurons were damaged and/or destroyed following MHV infection of the CNS. Immunohistochemical staining was performed on 8-¹M frozen sections using a mouse monoclonal antibody speci c for the neuron-speci c nuclear protein NeuN (1:500 dilution, Chemicon, Inc., Figure 3 Evidence of neuronal damage in MHV-infected NOS2 C=C mice. Shown are representative sections from MHV-infected NOS2 C=C and NOS2 ¡=¡ mice. Arrowheads indicate neurons (NeuN-positive cells) within the DG. Note the overall reduced thickness and disrupted pattern of the DG from infected NOS2 C=C mice as compared to the DG obtained from infected NOS2 ¡=¡ mice. Original magni cation, x200. Control sections with omission of primary antibody were negative. Sections from sham-infected mice treated with primary and secondary antibodies showed healthy neurons (not shown). Temecula, California). Diaminobenzidine was used for a chromagen and neuronal nuclei are stained brown. Such analysis revealed an overall reduction in the thickness and disrupted pattern within the dentate gyrus (DG) combined with the appearance of fragmented neuronal nuclei within the DG and hippocampus of infected NOS2 C=C mice (Figure 3 ). In marked contrast, neurons present within the hippocampus and DG of MHV-infected NOS2 ¡=¡ mice appeared normal with no apparent damage (Figure 3 ). In situ TUNEL analysis was combined with immuno uorescent staining to determine if neurons were undergoing apoptosis within the brains of MHV-infected NOS2 C=C and NOS2 ¡=¡ mice. Confocal microscopy (Bio-Rad MRC UV laser-scanning confocal microscopy, Bio-Rad, Richmond, California) was performed on ethanol-xed frozen brain sections (8 ¹M) obtained from MHV-infected NOS2 C=C and NOS2 ¡=¡ mice at day 7 p.i. For visualization of NeuNpositive cells, a TRITC-labeled secondary antibody was used (1:50, Sigma, St. Louis, Missouri). TUNELpositive cells were detected through use of FITCconjugated enzyme TdT (Boehringer Mannheim). Such analysis revealed that >70% of TUNEL-positive cells present in NOS2 C=C mice were neurons suggesting that these cells were apoptotic (Figure 4, Table 2 ). In contrast, only limited numbers of TUNEL-positive cells were seen within the brains of NOS2 ¡=¡ mice and less than 5% were neurons (Figure 4, Table 2 ). Sham mice of both groups showed limited TUNELstaining. Neither macrophage/microglia nor astrocytes were found to be a major source of apoptotic cells within the brains of infected mice (data not shown). The ndings presented indicate that NOS2generated NO does not contribute to demyelination in MHV-infected mice. These data are consistent with a recent study by Wu et al (2000) that indicated that NOS2 is not required for demyelination to occur following MHV infection of the CNS. In addition, Oleszak et al (1997) have reported that NOS2-derived NO does not participate in demyelination within mice infected with Theiler's murine encephalomyeli-tis virus (TMEV). Therefore, the collective evidence indicates that NOS2 expression is not important in contributing to demyelination in two separate viral models of the human demyelinating disease multiple sclerosis (MS). However, the results presented are in slight contrast with a recent report that indicated that NOS2-generated NO exhibited a transient effect on leukocyte in ltration and demyelination within the CNS of MHV-infected mice that correlated with a marked decrease in expression of mRNA transcripts for the proin ammatory chemokines CCL5 and CCL2 (Lane et al, 1999) . Collectively, these results suggested NOS2 activity was important in the induction phase of disease, in part, by regulating chemokine expression but did not substantially contribute to the chronic stage of disease. It should be emphasized that a Data are presented as mean § SEM. b n D number of elds counted. Enumeration of dual-positive cells present within the DG and hippocampus was performed on brain sections from infected mice at day 7 p.i. from staining shown in Figure 4 . A minimum of two mice per condition were counted. Sham brain sections showed less than 1% of the cells to be TUNELpositive. ¤ P · 0.001. these studies were performed in MHV-infected mice treated with the NOS2 inhibitor AG (Lane et al, 1999) . We have not found any evidence of signi cantly altered chemokine gene expression within the CNS of MHV-infected NOS2 ¡=¡ mice, suggesting that AG treatment may have a direct effect on chemokine transcription. In support of this is preliminary data indicating that AG treatment of cultured macrophages directly modulates chemokine gene expression (Chen and Lane, unpublished observations). Therefore, the results presented in the current study clearly indicate that NOS2-derived NO does not affect leukocyte entry into the CNS (data not shown) nor the induction of demyelination early following infection (day 12 p.i.) or at later times (day 35 p.i.) ( Table 2) . Expression of NOS2 has been associated with virus-induced neuropathology in several systems including borna virus (Koprowski et al, 1993; Akaike et al, 1995) , HIV (Raber et al, 1996) , LCMV (Campbell, 1996) , and HSV-1 (Meyding-Lamade et al, 1998) . Although the mechanisms involved in NOS2-mediated pathology within these different systems are not clear, it is possible that NO exerts a cytotoxic effect on resident cells of the CNS. Our data support a role for NOS2-derived NO in contributing to virus-induced neurologic disease by triggering events that lead to neuronal apoptosis. In support of this observation is a recent study by Rose et al (1998) that reported a signi cant reduction in apoptosis within the CNS in TMEV-infected mice treated with the NOS inhibitor AG. It is interesting to note that Wu et al (2000) did not report any differences in the severity of clinical disease between NOS2 C=C and NOS2 ¡=¡ mice following infection with MHV, which is marked contrast to the results presented in the current report. However, that study was performed with MHV strain J2-2-V1, which triggers a mild encephalomyelitis (in which the majority of mice survive) characterize d by sparing of neurons and replication in glial cells. In contrast, the virus used in the present studies (MHV-V5A13.1) is capable of replicating within both neurons and glia during acute disease and results in more severe encephalomyelitis and clinical disease. Therefore, the differences between the present study and Wu et al (2000) may be the result of viral strain differences and tropism for neurons. The data presented support previous studies that have clearly shown that NO is capable of inducing death in neurons Zhang et al, 1994; Keane et al, 1997; Melkova et al, 1997; Tamatani et al, 1998; Yuan and Yankner, 2000) . NO may trigger apoptosis by a variety of mechanisms including directly damaging DNA as well as inhibiting expression of the anti-apoptotic protein Bcl-2 (Nguyen et al, 1992; Mebmer et al, 1996; Antonsson et al, 1997; Dimmeler and Zeiher, 1997; Keane et al, 1997; Tamatani et al, 1998; Allione et al, 1999; Yuan and Yankner, 2000) . The mechanism(s) by which NO induces apoptosis in neurons following MHV infection are unknown and are the focus of ongoing study. 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