By
From the * Host Defense Laboratory, Viral Engineering and Cytokines Group, Division of
Immunology and Cell Biology, The John Curtin School of Medical Research, The Australian National
University, Canberra, ACT 2601, Australia; and the Beatrice and Samuel A. Seaver Laboratory,
Department of Medicine, Cornell University Medical College, New York 10021
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Abstract |
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Viral infection often activates the interferon (IFN)--inducible gene, nitric oxide synthase 2 (NOS2). Expression of NOS2 can limit viral growth but may also suppress the immune system and damage tissue. This study assessed each of these effects in genetically deficient NOS2
/
mice after infection with influenza A, a virus against which IFN-
has no known activity. At
inocula sufficient to cause consolidating pneumonitis and death in wild-type control mice,
NOS2
/
hosts survived with little histopathologic evidence of pneumonitis. Moreover, they
cleared influenza A virus from their lungs by an IFN-
-dependent mechanism that was not evident in wild-type mice. Even when the IFN-
-mediated antiviral activity was blocked in NOS2
/
mice with anti-IFN-
mAb, such mice failed to succumb to disease. Further evidence that this protection was independent of viral load was provided by treating NOS2+/+
mice with the NOS inhibitor, N
-methyl-L-arginine (L-NMA). L-NMA prevented mortality
without affecting viral growth. Thus, host NOS2 seems to contribute more significantly to the
development of influenza pneumonitis in mice than the cytopathic effects of viral replication.
Although NOS2 mediates some antiviral effects of IFN-
, during influenza infection it can
suppress another IFN-
-dependent antiviral mechanism. This mechanism was observed only
in the complete absence of NOS2 activity and appeared sufficient to control influenza A virus
growth in the absence of changes in cytotoxic T lymphocyte activity.
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Introduction |
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The discovery that the IFN--inducible nitric oxide synthase 2 (NOS2) gene could confer potent antiviral activity (1, 2) substantially extended its role as a critical component of the innate immune system (3). Yet although a
number of viruses are susceptible to the nitric oxide (NO)-
derived products of NOS2, not all are inhibited (3, 4, and
references therein). Moreover, as a broadly reactive, cell-permeant radical, NO has the potential to injure uninfected
cells near those harboring virus (3-7, and references
therein). For this reason, NOS2 has also been implicated in
the pathology associated with several viral models (4, 5). That most of the studies mentioned above have relied on
the use of NOS inhibitors of limited isoform specificity
complicates interpretation of disparate results, since all three
NOS isoforms can exhibit antiviral activity, and at least two
of them (NOS2 and NOS1) can damage cells (3, 4, 8, 9).
We have resorted to NOS2 gene-targeted mice (10) in
an effort to assess the contribution made by this locus towards pathogenesis and immunoregulation during viral infection. Such mice have confirmed NOS2's ability to protect the host during infection by Coxsackie B3 (11) and
ectromelia viruses (11a). Here we studied infection by
pneumotropic influenza A virus for two reasons. First, an
earlier report with pharmacologic inhibitors implicated
NOS2 in host pathology during influenza pneumonitis (6).
Second, although IFN- has well-characterized antiviral
activity and is important for NOS2 induction in vivo (1),
control of influenza A virus does not seem to require IFN-
,
as evidenced by studies using anti-IFN-
antibodies (12, and G. Karupiah, unpublished observations) and IFN-
/
mice (13, and G. Karupiah, unpublished observations). Surprisingly, treatment with neutralizing antibody revealed that
IFN-
can control influenza A virus, but only when NOS2
is absent.
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Materials and Methods |
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Mice.
NOS2Virus Infection and Enumeration.
Influenza A virus (strain A/ PR/8/34) was directly instilled in 30 µl PBS into the nasal cavity under Avertin (1.5 ml/100 g) anesthesia, and titers were thereafter determined via serial dilutions of lung homogenates plated onto MDCK cell monolayers for detection of PFU (15). The limit of assay detection was 2.0 log10 PFU.Lung Immunocyte Isolation.
Leukocytes were isolated as outlined (12) from HBSS-perfused and aseptically excised lungs from groups of influenza virus-infected mice (n = 5 per group). Populations consisted of ~40-50% T lymphocytes (CD3+), 20-30% B lymphocytes (CD45R/B220+), and 10-12% macrophages (F4/ 80+) as determined by flow cytometry.Bronchoalveolar Lavage Fluid NOx Assay.
Bronchoalveolar lavage fluid (BALF) consisted of washings from intratracheal instillation of 1.0 ml PBS. Nitrite was measured by a diazotization assay (14) with a sensitivity of 4 µM after NO3Cytotoxicity Assays.
Influenza A virus-infected and uninfected EL-4 lymphoma cells were used as targets for measurement of anti-influenza A virus-specific, class I MHC-restricted CTL activity of lung parenchymal cells using standard 51Cr-release assays (16, 17).IFN- ELISA Analysis.
Histology.
Formalin-fixed, paraffin-embedded tissues were sectioned (5 µm) and stained with hematoxylin and eosin as outlined (18).Neutralization of NOS2 and IFN- In Vivo.
Statistical Analysis.
Unpaired Student's t tests at 95% confidence levels were performed using InStat software, Version 2.00 (GraphPad Software for Science, San Diego, CA). ![]() |
Results and Discussion |
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In previous
work, mice treated with anti-IFN- antibodies (12, and G. Karupiah, unpublished observations) or lacking IFN-
(13, and G. Karupiah, unpublished observations) responded to
influenza A virus similarly to control mice, even though
their capacity for NOS2 expression is known to be impaired (19, 20). Our findings in NOS2
/
mice not only
corroborated the implication that NOS2 is dispensable for
the control of influenza A virus in the lower respiratory tract, but also demonstrated that NOS2 is detrimental in
this setting. Thus, intranasal inoculation with the virulent
A/PR/8/34 strain led to rapid pneumonitis and early mortality in wild-type mice given 300, 400, or 500 hemagglutinin units (HAU) of virus. In contrast, NOS2
/
mice
started to succumb only at 600 HAU (Fig. 1 a).
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Influenza A virus titers in the lungs of D-NMA-treated
controls given 400 HAU of virus reached 5.6 ± 0.2 log10
PFU by day 6 after infection, whereas in NOS2/
mice
lung virus titers were below the limit of detection (<2.0 log10 PFU; Fig. 1 b). Surprisingly, L-NMA-treated wild-type mice harbored viral loads similar to their D-NMA-
treated littermates (Fig. 1 b) but, unlike the latter, they
showed no signs of morbidity and survived through the 21-d
period of observation (data not shown). This outcome
suggested (a) that NOS2
/
mice selectively expressed a
potent antiinfluenza effector mechanism, and (b) the high
output NO pathway may contribute to pathogenesis, since
both L-NMA- and D-NMA-treated wild-type mice failed
to eradicate the virus but only those given the inactive
D-enantiomer proved susceptible, dying between days 7 and 8 after infection, like their untreated counterparts (Fig.
1 a, and data not shown).
Experiments in 2-microglobulin
/
mice (21) and Fas
/
× perforin
/
chimeras (22) demonstrated the importance of CD8+ T lymphocytes for elimination of influenza A virus from the lung. To determine if
this effector arm of the immune response contributed to
rapid virus clearance in the NOS2
/
host, we examined
influenza-specific CTL activity on day 6 after infection.
Lung immunocytes lysed the influenza virus-infected targets equally well irrespective of NOS2 status (Fig. 2 a).
However, measurement of local IFN-
production by
these cells (Fig. 2 b) and in BALF (Fig. 2 c) revealed that
NOS2
/
mice exhibited 1.9-3.4-fold higher levels of
IFN-
than wild-type hosts; this increase was independent
of antigen load (Fig. 1 b).
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Whether such an increase in IFN- secretion could account for the reduced viral burden in mutant animals was
assessed by intraperitoneal injection with mAb to IFN-
(18). Assay of virus infectivity levels 6 d after infection
demonstrated that IFN-
-dependent anti-influenza A virus
mechanisms were operative in mutant mice as depicted by the high titers comparable to those of L-NMA or D-NMA-
treated wild-type controls (Fig. 2 d). In contrast, NOS2
/
animals receiving an IgG1 isotype-matched control mAb
still retained their capacity to restrict influenza virus replication within the lungs; titers were below the level of detection (Fig. 2 d).
The demise of untreated or D-NMA-treated NOS2+/+ mice was
not due to uncontrolled viral growth, since L-NMA-
treated wild-type or anti-IFN--treated NOS2
/
hosts
also displayed overwhelming lung viral burdens yet showed no signs of morbidity throughout the 3-wk experimental
period (Figs. 1 b, 2 d, and data not shown). Survival of the
latter two groups implied that pulmonary injury in D-NMA-
treated animals arose as a consequence of excessive NO
production rather than by direct viral cytopathic effects.
Indeed, NOx levels within the BALF of D-NMA-treated
NOS2+/+ mice rose appreciably (~3.4-4.0-fold) during
the course of infection (Fig. 3 a), whereas this increase was
absent in mutant mice and inhibited by 77-90% in L-NMA-
treated controls (Fig. 3 a). Histopathologic examination
revealed massive inflammatory foci and edema within the
lungs of all D-NMA-treated wild-type mice as part of the
consolidating pneumonitis observed 6 d after infection
(Fig. 3 b). Neutrophils and alveolar macrophages were
especially prominent. Both are capable of releasing cytotoxic oxidants besides NO (6, 23), such as the superoxide
anion (O2
), which can react with the latter to yield
more damaging products like peroxynitrite (ONOO
[3]).
Akaike et al. have demonstrated the local formation of both
ONOO
and NO-hemoglobin in murine influenza infection using nitrotyrosine footprinting and electron spin resonance spectroscopy (6), while NO-thiol adducts also appear elevated in wild-type but not NOS2
/
mice during
tuberculosis (14). Thus, it is conceivable that several NO-derived species acted in concert to cause influenza-associated pathology.
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In marked contrast to the severe pneumonitis seen for all
D-NMA-treated NOS2+/+ mice, the majority of mutant
animals (~70%) lacked evidence of pathology. A mild degree of inflammation was observed in the remainder; an
example of the latter is depicted in Fig. 3 b. These results are consistent with the idea that NOS2 promotes the development of viral pneumonia. Additional support for
this concept comes from recent studies using NOS inhibitors during infection with HSV-1 (7), murine CMV
(24), or the influenza A/Kumamoto/Y5/67 (H2N2) mouse
adapted strain (6). Inhibition of NOS fully protected mice
from HSV-1-dependent lung injury despite a 17-fold increase in viral titers (7). That NO was necessary to control
HSV-1 in vivo substantiated earlier experiments in vitro (1,
2). In contrast, in the present work, the functional loss of
NOS2 alleviated pneumonitis and greatly improved survival irrespective of whether influenza virus was cleared
(NOS2/
hosts) or not (NOS2+/+ mice given L-NMA).
Reduced pathology has also recently been noted in NOS2
/
mice infected with Mycobacterium avium (25) and Toxoplasma gondii (26).
IFN- has been implicated as the major cytokine responsible for NOS2 induction during influenza A-induced
pneumonitis (6). Aside from IFN-
's purported proinflammatory action in wild-type mice, we found that heightened
IFN-
release was both associated with and required for influenza A virus clearance in NOS2
/
animals, an effect
unaccompanied by changes in antiviral CTL activity (Fig. 2,
a-d). The effect was specific for mutant mice, since L-NMA-
treated wild-type mice had comparable IFN-
levels but
failed to limit viral growth. IFN-
thus appears to cooperate with immune components otherwise subject to suppression by NOS2 and not by NOS1 and/or NOS3. Complete inhibition of NOS2 by disruption of the gene
encoding it, rather than the partial inhibition afforded by
treatment of NOS2+/+ mice with L-NMA, also appeared
necessary to permit expression of the IFN-
-dependent
antiinfluenza mechanism. Among pertinent candidates for
components of this mechanism are MHC class II expression by pulmonary dendritic cells (27) and Th1 cell proliferation plus cytokine production (28, 29). Each could be
compromised by NOS2, which can limit T cell responsiveness to IL-2 in the lungs of rats and mice (28) as well as inhibit the release of IL-12 and elevate the expression of
TGF-
as shown in Leishmania major-infected NOS2
/
mice (30). In this manner, the high output NO pathway
may regulate IFN-
secretion via a feedback mechanism,
such that once infection is cleared, autotoxic NO production is extinguished.
Beyond this, how NOS2 might influence the underlying
molecular events leading to cytokine gene expression and
action during antiviral immunity remains speculative. NO
can activate or disable protein tyrosine kinases of the Janus
kinase (JAK), extracellular signal-regulatory kinase (ERK),
and src families, as well as the transcription factor nuclear
factor (NF)-B, and thus may affect cytokine signaling (31, and references therein). Indeed, NOS2
/
animals have altered levels of NF-
B and signal transducer and activator of
transcription (STAT)3 activation in hemorrhage/resuscitation (32) and perturbed IFN-
release in L. major (28, 29)
and influenza A infections (Fig 2, b and c). Cytokine expression could also be regulated by NOS2 at the posttranslational level. For example, S-nitrosylation of the active site
cysteine residue in caspases leads to their inactivation (33).
If this inhibition were to be relieved in NOS2
/
mice, abnormally active caspase-1 might more readily promote the
release of IL-18 (34, 35), a powerful inducer of IFN-
.
During infection, NOS2 probably displays all three of its
major effects to variable degreesantimicrobial, inflammatory, and immunosuppressive. The results seen here with
influenza A virus-infected mice appear to illustrate one extreme, in which the latter two effects dominate.
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Footnotes |
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Address correspondence to Gunasegaran Karupiah, Division of Immunology and Cell Biology, The John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. Phone: 61-2-6249-2627; Fax: 61-2-6249-2595; E-mail: guna.karupiah{at}anu.edu.au
Received for publication 13 February 1998 and in revised form 26 June 1998.
We thank Drs. Nicole Baumgarth and Anne Kelso for lung parenchymal cell isolation methods.
This work was supported by the National Centre for HIV Research (G. Karupiah and J.-H. Chen), and by National Institutes of Health grants HL-51967 and AI-34543 (to C.F. Nathan).
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