Both inducible nitric oxide synthase and NADPH oxidase contribute to the control of virulent phase I Coxiella burnetii infections

Host control of Coxiella burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The activation of macrophages by cytokines leads to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that have potent antimicrobial activities. The contributions of ROI and RNI to the inhibition of C. burnetii replication were examined in vitro by the use of murine macrophage-like cell lines and primary mouse macrophages. A gamma interferon (IFN-gamma) treatment of infected cell lines and primary macrophages resulted in an increased production of nitric oxide (NO) and hydrogen peroxide (H2O2) and a significant inhibition of C. burnetii replication. The inhibition of replication was reversed in the murine cell line J774.16 upon the addition of either the inducible nitric oxide synthase (iNOS) inhibitor NG-monomethyl-L-arginine (NGMMLA) or the H2O2 scavenger catalase. IFN-gamma-treated primary macrophages from iNOS-/- and p47phox-/- mice significantly inhibited replication but were less efficient at controlling infection than IFN-gamma-treated wild-type macrophages. To investigate the contributions of ROI and RNI to resistance to infection, we performed in vivo studies, using C57BL/6 wild-type mice and knockout mice lacking iNOS or p47phox. Both iNOS-/- and p47phox-/- mice were attenuated in the ability to control C. burnetii infection compared to wild-type mice. Together, these results strongly support a role for both RNI and ROI in the host control of C. burnetii infection.     

Relaxation by vasoactive intestinal polypeptide in the gastric fundus of nitric oxide synthase-deficient mice

In many gastrointestinal tissues nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) both play a role as inhibitory non-adrenergic non-cholinergic neurotransmitters. As the mode of interaction between NO and VIP remains controversial, the aim of this study was to investigate the interplay between NO and VIP in the mouse gastric fundus and to evaluate the nitric oxide synthase (NOS) isoform involved in VIP-induced relaxation by using inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) knockout mice. The influence of NOS inhibitors on the relaxant effect of VIP was determined in isolated smooth muscle cells and smooth muscle strips of wild-type and knockout mice. In isolated smooth muscle cells from wild-type, eNOS knockout and nNOS knockout mice, the relaxation induced by VIP (10⁻⁹ m ) was inhibited by approximately 70–95 % by both the non-selective NOS inhibitor N^G -nitro- l -arginine ( l -NA; 10⁻⁴ m ) and the selective inducible NOS inhibitor N -(3-(aminomethyl)-benzyl)acetamidine (1400W; 10⁻⁶ m ). In cells isolated from iNOS knockout mice, VIP still induced full relaxation but it was not influenced by l -NA or 1400W. In smooth muscle strips from wild-type and knockout mice, the concentration-dependent relaxation by VIP (10⁻⁹ to 3 × 10⁻⁷ m ) was not influenced by l -NA or 1400W. These results suggest that the experimental method determines the influence of NOS inhibitors on the relaxant effect of VIP. iNOS, probably induced by the isolation procedure, might be involved in the relaxant effect of VIP in isolated smooth muscle cells but not in classic smooth muscle strips.     

A critical role for inducible nitric oxide synthase in host survival following coxsackievirus B4 infection

Coxsackieviral infections have been linked etiologically to multiple diseases. The serotype CB4 is associated with acute pancreatitis and autoimmune type 1 diabetes. To delineate the mechanisms of host survival after an acute infection with CB4 (strain E2), we have investigated the role of nitric oxide (NO), generated by the inducible form of nitric oxide synthase (NOS2), in viral clearance and pancreatic beta-cell maintenance. Mice deficient in NOS2 (NOS2-/- mice) and their wild-type (wt) counterparts were injected with CB4, after which both groups developed severe pancreatitis, hepatitis, and hypoglycemia within 3 days. Within 4 to 7 days postinfection (p.i.), most of the NOS2-/- mice died and at a strikingly higher mortality rate than wt mice. Histological examination of pancreata from both infected NOS2-/- and infected wt mice revealed early and complete destruction of the pancreatic acinar tissue, but intact, insulin-stained islets. When examined up to 8 weeks p.i., neither surviving NOS2-/-mice nor surviving wt mice developed hyperglycemia. However, the clearance of infectious CB4 was different between the mice. The spleens of NOS2-/- survivors were cleared of infectious virus with kinetics similar to that of wt mice, but the livers, pancreata, kidneys, and hearts of the NOS2-/- groups cleared virus more slowly than those of the wt group. This delayed clearance was particularly prominent in the livers of infected NOS2-/- mice, which also showed prolonged histopathological features of viral hepatitis. Taken together, this outcome suggests that NOS2 (and NO) is not required for the prevention of pancreatic beta-cell depletion after CB4 infection. Instead the critical actions of NOS2 apparently occur early in the host immune response, allowing mice to survive and clear virus. Moreover, the data support the existence of an organ-specific dependency on NO for a rapid clearance of CB4.     

Induced production of nitric oxide and sensitivity of alveolar macrophages derived from mice with different sensitivity to Coxiella burnetii

We compared in vitro sensitivities to Coxiella burnetii of alveolar macrophages, derived from mice sensitive and resistant to C. burnetii, respectively, and examined the role of nitric oxide (NO) in the C. burnetii infection. Alveolar macrophages of sensitive A/J mice showed a larger population of C. burnetii antigen-positive cells than those of resistant C57BL/6 mice. C. burnetii induced NO production in alveolar macrophages, but N-methyl-L-arginine and sodium nitroprusside (SNP), NO inhibitor and donor, respectively, did not inhibit the infection. Thus the NO induction seems to be independent of the cell defense mechanism against the C. burnetii infection.     

Role for inducible nitric oxide synthase in protection from chronic Chlamydia trachomatis urogenital disease in mice and its regulation by oxygen free radicals.

It has been previously reported that although inducible nitric oxide synthase (iNOS) gene knockout (NOS2(-/-)) mice resolve Chlamydia trachomatis genital infection, the production of reactive nitrogen species (RNS) via iNOS protects a significant proportion of mice from hydrosalpinx formation and infertility. We now report that higher in vivo RNS production correlates with mouse strain-related innate resistance to hydrosalpinx formation. We also show that mice with a deletion of a key component of phagocyte NADPH oxidase (p47(phox-/-)) resolve infection, produce greater amounts of RNS in vivo, and sustain lower rates of hydrosalpinx formation than both wild-type (WT) NOS2(+/+) and NOS2(-/-) controls. When we induced an in vivo chemical block in iNOS activity in p47(phox-/-) mice using N(G)-monomethyl-L-arginine (L-NMMA), a large proportion of these mice eventually succumbed to opportunistic infections, but not before they resolved their chlamydial infections. Interestingly, when compared to WT and untreated p47(phox-/-) controls, L-NMMA-treated p47(phox-/-) mice resolved their infections more rapidly. However, L-NMMA-treated p47(phox-/-) mice lost resistance to chronic chlamydial disease, as evidenced by an increased rate of hydrosalpinx formation that was comparable to that for NOS2(-/-) mice. We conclude that phagocyte oxidase-derived reactive oxygen species (ROS) regulate RNS during chlamydial urogenital infection in the mouse. We further conclude that while neither phagocyte oxidase-derived ROS nor iNOS-derived RNS are essential for resolution of infection, RNS protect from chronic chlamydial disease in this model.     

Nitric oxide exerts distinct effects in local and systemic infections with Streptococcus pneumoniae

Nitric oxide (NO) is known to be involved in the immune response against a range of organisms. Little is known about the effects of nitric oxide in pneumococcal infections. We have now investigated the role of nitric oxide in local and systemic infections caused by Streptococcus pneumoniae in NOS2 deficient mice. Although a deficiency in NO does not affect survival of mice during pneumococcal pneumonia, NO does control pneumococcal viability within the lung airways and tissue. Bronchoalveolar lavage fluid (BALF) from NOS2-deficient mice contained significantly elevated TNF activity, IFNgamma and total protein during mid/late infection. Incubation of S. pneumoniae with the NO donor SNAP revealed a direct anti-pneumococcal effect for NO in vitro. Deficiency in NOS2 did not affect bacteraemia following intranasal infection. In contrast NOS2-deficient mice were significantly less susceptible to intravenous infection with S. pneumoniae than were wild type mice and were able to control pneumococcal viability within the bloodstream. Our results indicate that NO is required within the lungs for anti-bacterial activity during the pneumococcal pneumonia but during Gram-positive bacteraemia NO is associated with increased bacterial loads and reduced survival.     

Depressed immune functions in the early phase of varicella-zoster virus reactivation

Severe combined immune deficient (SCID) mice inoculated intravenously with vaccinia virus (VV) became sick within 6–8 days and died 10–12 days after infection. Tail lesions developed and the number depended on the virus inoculum. Age-matched immunocompetent NMRI mice similarly infected also developed tail lesions but did not become sick. When the infected SCID mice were treated with the acyclic nucleoside phosphonate HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine], either for 5 consecutive days starting on the day of infection or for 5 consecutive days starting on day 2, 4, or 6 post infection, or as a single dose at 7 days or 1 day before infection, VV-associated death was significantly delayed. VV-infected SCID mice that received two doses of 20 mg/kg of HPMPC every week survived the infection for about 130 days. The period during which the mice remained disease-free following HPMPC treatment correlated with the absence of detectable virus in their organs. The VV/SCID mouse model employed here may be useful for determining whether (attenuated) recombinant VV (carrying HIV genes) may have detrimental effects in the immunodeficient host. HPMPC may be considered as a drug candidate for the treatment and prophylaxis of such complications.     

Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1(+))CD11b(+ )immature myeloid suppressor cells

Trypanosoma cruzi infection is associated with a severe unresponsiveness of spleen cells (SC) to antigens and mitogens. A high production of NO by concanavalin A (Con A)-stimulated SC from infected but not from control mice was observed. Neutralization of endogenous IFN-gamma production or treatment with NO synthase (NOS) inhibitor, L-N-monomethyl-arginine, blocked Con A-induced NO production and greatly restored proliferation by SC from infected mice. This was confirmed by using IFN-gammaR(-/-) and inducible NOS (iNOS)(-/- )knockout mice, since unresponsiveness to mitogens of SC from those infected mice was much less pronounced than in control littermates. Interestingly, SC unresponsiveness was associated with a huge increase in CD11b(+) cells that express Ly-6G (Gr1)(+) and other immature myeloid markers These cells were absent in infected IFN-gammaR(-/-) spleens. Purified immature Gr1(+)CD11b(+) cells produced NO and expressed iNOS upon IFN-gamma treatment, and were able to inhibit T cell proliferation. In addition, depletion of myeloid CD11b(+ )cells abrogated NO production and restored mitogen-induced proliferation, but not IL-2 synthesis, in SC from infected mice. IL-2 production and CD25 cell surface expression by mitogen-activated T cells were greatly depressed in SC from IFN-gammaR(-/-) and iNOS(-/- )mice, confirming that Gr1(+)CD11b(+) cells were not involved in their down-regulation. In contrast, IL-5, tumor necrosis factor and IFN-gamma production, and CD69 expression by T cells were not depressed in infected SC. The results indicate the existence of an immunosuppressive mechanism during T. cruzi infection, mediated through IFN-gamma-dependent NO secretion by immature Ly-6G (Gr1)(+)CD11b(+ )myeloid cells.     

Spontaneous phthiocerol dimycocerosate-deficient variants of Mycobacterium tuberculosis are susceptible to gamma interferon-mediated immunity

Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-γ)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2(-/-) mice but were growth-proficient and virulent in IFN-γ(-/-) mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-γ-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2(-/-) and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-γ-mediated immune response that is independent of NOS2.     

Skin inflammation in RelB(-/-) mice leads to defective immunity and impaired clearance of vaccinia virus

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disorder occurring in genetically predisposed individuals with a systemic T(H)2 bias. Atopic dermatitis patients exposed to the smallpox vaccine, vaccinia virus (VV), occasionally develop eczema vaccinatum (EV), an overwhelming and potentially lethal systemic infection with VV. OBJECTIVE: To establish a murine model of EV and examine the effects of skin inflammation on VV immunity. METHODS: The skin of RelB(-/-) mice, like that of chronic AD lesions in humans, exhibits thickening, eosinophilic infiltration, hyperkeratosis, and acanthosis. RelB(-/-) and wild-type (WT) control mice were infected with VV via skin scarification. Viral spread, cytokine levels, IgG2a responses and VV-specific T cells were measured. RESULTS: Cutaneously VV-infected RelB(-/-), but not WT mice, exhibited weight loss, markedly impaired systemic clearance of the virus and increased contiguous propagation from the inoculation site. This was associated with a dramatically impaired generation of IFN-gamma-producing CD8(+) vaccinia-specific T cells along with decreased secretion of IFN-gamma by VV-stimulated splenocytes. The T(H)2 cytokines-IL-4, IL-5, IL-13, and IL-10-on the other hand, were overproduced. When infected intraperitoneally, RelB(-/-) mice generated robust T cell responses with good IFN-gamma production. CONCLUSION: Allergic inflammation in RelB(-/-) mice is associated with dysregulated immunity to VV encountered via the skin. We speculate that susceptibility of AD patients to overwhelming vaccinia virus infection is similarly related to ineffective T cell responses. CLINICAL IMPLICATIONS: The susceptibility of patients with AD to EV following cutaneous contact with VV is related to ineffective antiviral immune responses.     

Host genetic background determines whether IL-18 deficiency results in increased susceptibility or resistance to murine Leishmania major infection

Interleukin-18 (IL-18) plays an important role in innate and acquired immunity. IL-18 gene deficient (IL-18-/-) mice of the 129 x CD1 strain were reported to be more susceptible to Leishmania major infection than the wild-type mice. In contrast IL-18-/- mice of the C57BL/6 background were found to be as resistant as the wild-type (WT) mice. To resolve this discrepancy, IL-18 gene deficiency was introduced by backcrossing on to the highly susceptible BALB/c, or the moderately resistant DBA/1 backgrounds. Here we have demonstrated that BALB/c IL-18-/- mice were more resistant to L. major infection than WT BALB/c mice, whereas DBA/1 IL-18-/- mice were markedly more susceptible than their WT littermates. BALB/c IL-18-/- mice produced less IFNgamma and IL-4, whereas DBA/1 IL-18ko mice produced more IFNgamma and IL-4 than their respective WT controls. These result clearly demonstrate that the role of IL-18 in resistance or susceptibility to L. major is determined by host genetic background.     

Endogenous nitric oxide attenuates erythropoietin gene expression in vivo

This study aimed to investigate the role of endogenous nitric oxide (NO) in erythropoietin (EPO) gene expression in mice in vivo. For this purpose EPO mRNA was semiquantitated by ribonuclease protection assay in livers and kidneys of three groups of mice: wild-type (wt), endothelial NO-synthase (NOS) knockout mice (eNOS-/-), and wt treated with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (50 mg x kg(-1) x day(-1)) for 4 days (wt+L-NAME). EPO gene expression was stimulated by normobaric hypoxia (8% O2) or by 0.1% carbon monoxide (CO) inhalation for 4 h each, or by intraperitoneal injection of 60 mg/kg cobaltous chloride (CoCl2) for 6 h. Renal EPO mRNA in wt increased 12-, 40-, and 13-fold over normoxic levels in response to hypoxia, CO and CoCl2 respectively. EPO mRNA was detectable in the livers only after CO exposure. Renal and hepatic EPO gene expression in wt+L-NAME appeared moderately increased relative to wt with a maximal 2.5-fold enhancement after CO exposure. EPO mRNA levels in eNOS-/- mirrored those of wt+L-NAME, but the effects were less prominent. Our data suggest that endogenous NO attenuates EPO gene expression in mice. This effect is dependent on the rate of EPO gene induction.     

Role for the CD28 molecule in the control of Coxiella burnetii infection

Q fever is an infectious disease caused by Coxiella burnetii, an obligate intracellular bacterium that replicates in macrophages. As cell-mediated immune response to microbial pathogens requires signals mediated by T-cell receptors and costimulatory molecules such as CD28, we wondered if CD28 is involved in protection against C. burnetii infection. CD28-deficient (CD28-/-) mice were inoculated with C. burnetii by intraperitoneal and intravenous routes. With both wild-type and CD28-/- mice, C. burnetii organisms were detected exclusively in spleen and liver. The antibody response against C. burnetii was impaired in CD28-/- animals, but, surprisingly, the lack of CD28 decreased C. burnetii burden in the infected tissues, whatever the manner of inoculation of bacteria. The CD28 deficiency had no effect on either granuloma formation, which reflects cell-mediated immunity against C. burnetii, or the production of gamma interferon and tumor necrosis factor, two cytokines known to be involved in granuloma formation. On the other hand, the production of interleukin-10 (IL-10) by peritoneal macrophages was highly impaired in CD28-/- mice. The results suggest that CD28 initiates a signal that favors C. burnetii replication through the modulation of the IL-10 pathway.     

Neuronal expression of NOS-1 is required for host recovery from viral encephalitis.

The role of nitric oxide synthase (NOS) in host defense and clearance of vesicular stomatitis virus (VSV) from the central nervous system (CNS) was examined. NOS-1, NOS-2, and NOS-3 knockout mice were infected with VSV and were treated with either IL-12 or medium. IL-12 treatment resulted in substantially decreased VSV titers in wildtype and NOS-3 knockout mice, but had a marginal effect in the NOS-1 and NOS-2 knockout mice. NOS-1 expression in neurons was associated with survival from VSV infection. The data indicate that the enzyme activity is local, since NOS-2 expression in microglia and inflammatory macrophages and NOS-3 expression in astrocytes, endothelial cells, and ependymal cells did not compensate.     

CD40/CD40 ligand interactions in the host defense against disseminated Candida albicans infection: the role of macrophage-derived nitric oxide

CD40L interaction with CD40 is required for normal cellular immune responses such as T cell-mediated activation of monocytes/macrophages, proinflammatory cytokine production, and leukocyte extravasation. We investigated the role of CD40/CD40 ligand (L) interactions during disseminated candidiasis in CD40L knockout (CD40L-/-) mice. While early during infection there were no differences in the Candida albicans outgrowth in the organs of wild-type and knockout mice, the CD40L-/- mice had a significantly increased yeast load in the kidneys compared to CD40L+/+ mice late during infection. Similar effects were observed in CD40L+/+ mice in which CD40 ligation was blocked by a neutralizing anti-CD40 antibody. The peak TNF-alpha plasma concentrations were significantly lower in the CD40L-/- mice than in CD40L+/+ mice. C. albicans-stimulated production of nitric oxide (NO) by peritoneal macrophages from CD40L-/- in vitro was significantly lower than that of control mice, and this was responsible for a reduced candidacidal activity of CD40L-/- macrophages. The role of endogenous NO synthesis induced by CD40 ligation for the defense against disseminated candidiasis was further demonstrated by the absence of these effects in knockout mice deficient in inducible NO-synthase. In conclusion, absence of CD40/CD40L interactions results in increased susceptibility to disseminated infection with C. albicans through decreased NO-dependent killing of Candida by macrophages.     

Differential roles of neuronal and endothelial nitric oxide synthases during carrageenan-induced inflammatory hyperalgesia

The present study investigated the role of neuronal nitric oxide synthase (nNOS) in carrageenan-induced inflammatory pain by combining genomic and pharmacological strategies. Intrathecal injection of the nNOS inhibitor 7-nitroindazole dose-dependently inhibited carrageenan-induced thermal hyperalgesia in both early and late phases in wild-type mice. However in nNOS knockout mice, carrageenan-induced thermal hyperalgesia remained intact in the early phase but was reduced in the late phase. Spinal Ca2+ -dependent nitric oxide synthase (NOS) activity in nNOS knockout mice was significantly lower than that in wild-type mice. Following carrageenan injection, although the spinal Ca2+ -dependent NOS activity in both wild-type and knockout mice increased, the enzyme activity in nNOS knockout mice reached a level similar to that in wild-type mice. On the other hand, no significant difference in spinal Ca2+ -independent NOS activity was noted between wild-type and nNOS knockout mice before and after carrageenan injection. Furthermore, intrathecal administration of the endothelial NOS (eNOS) inhibitor L-N5-(1-iminoethyl)-ornithinein nNOS knockout mice inhibited the thermal hyperalgesia in both early and late phases, though this inhibitor had no effect in wild-type mice. Meanwhile, Western blot showed that eNOS expression in the spinal cord of nNOS knockout mice was up-regulated compared with wild-type mice; immunohistochemical staining showed that the spinal eNOS was mainly distributed in superficial laminae of the dorsal horn. Finally, double staining with confocal analysis showed that the enhanced spinal eNOS was expressed in astrocytes, but not in neurons. Our current results indicate that nNOS plays different roles in the two phases of carrageenan-induced inflammatory pain. In this model, enhanced spinal eNOS appears to compensate for the role of nNOS in nNOS knockout mice.     

Role of interferon and interferon regulatory factors in early protection against Venezuelan equine encephalitis virus infection

To investigate the role of type I interferon (IFN) and its regulatory transacting proteins, interferon regulatory factors (IRF-1 and IRF-2), in early protection against infection with virulent Venezuelan equine encephalitis virus (VEE), we utilized mice with targeted mutations in the IFN-alpha/beta receptor, IRF-1, or IRF-2 genes. IFN-alpha/beta-receptor knockout mice are highly susceptible to peripheral infection with virulent or attenuated VEE, resulting in their death within 24 and 48 h, respectively. Treatment of normal macrophages with anti-IFN-alpha/beta antibody prior to and during infection with molecularly cloned virulent VEE resulted in increased VEE replication. However, treatment with high doses of IFN or IFN-inducing agents failed to alter percentage mortality or average survival times in mice challenged with a low dose of virulent VEE. In IRF-1 and IRF-2 knockout mice (IRF-1(-/-) and IRF-2(-/-)), the 100% protection against virulent VEE that is conferred by attenuated VEE within 24 h in control C57BL/6 mice was completely absent in IRF-2(-/-) mice, whereas 50% of IRF-1(-/-) mice were protected. IRF-2(-/-) mice were deficient in clearing VEE virus from the spleen and the brain compared to the heterozygous IRF-2(+/-) knockout or C57BL/6 (+/+) mice. Furthermore, a distinct pattern of histopathological changes was observed in brains of IRF-2(-/-) mice after VEE exposure. Taken together, these findings imply that the altered immune response in IRF-1 and IRF-2 knockout mice results in altered virus dissemination, altered virus clearance, and altered virus-induced pathology. Thus, type I interferon, as well as IRF-1 and IRF-2, appears to play an important and necessary role in the pathogenesis of, and protection against, VEE infection.     

Role of interferon gamma in the pathogenesis of primary respiratory syncytial virus infection in BALB/c mice

Immunologic mechanisms are thought to contribute to the pathogenesis of respiratory syncytial virus (RSV) bronchiolitis in humans. RSV-infected BALB/c mice exhibit tachypnea and signs of outflow obstruction, similar to symptoms in humans. Interferon gamma (IFNgamma) has been found to be the predominant cytokine produced in humans and mice with RSV infection. We therefore undertook this study to evaluate the role of IFNgamma in the development of respiratory illness in RSV-infected mice. BALB/c mice were infected with RSV, and lung function was assessed by plethysmography. Bronchoalveolar lavage (BAL) fluids were analyzed for the concentration of interferon gamma (IFNgamma) and the presence of inflammatory cells, and lung tissue sections were examined for histopathologic changes. The role of IFNgamma was further addressed in studies of IFNgamma knock-out mice (IFNgamma(-/-)) and of mice depleted of IFNgamma by in vivo administration of a neutralizing antibody. After infection, mice developed respiratory symptoms that were strongly associated with the number of inflammatory cells in BAL, as well as with the concentrations of IFN-gamma. Both IFN-gamma(-/-) mice and mice treated with anti-IFNgamma developed more extensive inflammation of the airways than control mice. However mice lacking IFNgamma exhibited less severe signs of airway obstruction. Together these data suggest a protective role of IFNgamma in RSV infection in terms of limiting viral replication and inflammatory responses but also a pathogenic role in causing airway obstruction.     

Strain-dependent requirement for IFN-γ for respiratory control and immunotherapy in murine gammaherpesvirus infection

Interferon-γ (IFN-γ) and perforin (pfp) are important effector mechanisms used by CD8 T cells to clear virus-infected cells. In this study, we used IFN-γ/pfp double knockout mice to address if these two effector molecules play redundant roles in the control of acute infection with murine gammaherpesvirus-68 (MHV-68) in BALB/C mice. Perforin knockout (KO) mice and wild-type mice cleared infectious virus from the lungs, even following high-dose infection. However, the IFN-γ KO and IFN-γ/pfp double knockout (DKO) groups had higher virus titers in the lungs at day 10 post-infection, and both groups had higher mortality rates. In IFN-γ/pfp DKO mice, the virus titer and mortality rate were significant higher than in IFN-γ KO mice, indicating a role for perforin in protection from disease. WT mice given IFN-γ blocking antibody also showed significantly higher viral titers. In contrast, IFN-γ KO mice on a C57BL/6 background controlled respiratory infection comparably to wild-type mice. These data show that perforin plays a redundant role in the control of virus replication, but IFN-γ plays an essential role in BALB/C mice infected with MHV-68. We conclude that there is a marked strain-dependent difference in the effector mechanisms needed to control acute MHV-68 infection between C57BL/6 and BALB/C mice. In addition we show that immune therapy that re-establishes viral control after spontaneous reactivation in CD4-deficient mice depends upon perforin in C57BL/6 mice but IFN-γ in BALB/C mice.