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Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA 94720
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Epidemiologic and in vitro data suggest that DHF/DSS may be an immunopathogenic disease (2, 3, 4, 5). However, the precise role of the immune system in response to DEN infection in vivo is not fully defined, in part due to the lack of an adequate animal model for DEN infection and disease. Therefore, to investigate the immune response to DEN infection in vivo as well as to begin to develop a better animal model for DEN infection and disease, we have optimized a mouse model of primary DEN infection (6). Based on this mouse model, we have recently demonstrated that the combined receptor activities of both IFN-
and IFN-
are essential and are more important than T and B cell-mediated responses in controlling primary DEN infection in vivo (7). Specifically, 129/Sv/Ev (129) mice lacking receptors for both IFN- and IFN- (IFN-R–/– x IFN-R–/– 129/Sv/Ev (AG129)) exhibit paralysis and die due to primary DEN infection, whereas T and B cell-deficient RAG1–/– C57BL/6J or RAG2–/– 129 mice can resolve acute DEN infection. Based on these results, we are now investigating the mechanism of IFN action in mice with primary DEN infection.
A hallmark feature of the IFN system in viral infections is a highly coordinated production of IFN-, involving several proteins from the IFN regulatory factor (IRF) family (8). In the early phase, constitutively expressed IRF3 induces the production of IFN-, which, in turn, induces IRF7. IRF3 and IRF7 then collectively initiate and amplify IFN- production. During RNA virus infections, IRF3 may be activated via the TLR-dependent and -independent pathways (9, 10). The TLR-dependent pathway is likely to involve the TLR3-Toll/IL-1R domain-containing adaptor inducing IFN- or TLR7/8-MyD88 signaling cascades, whereas the TLR-independent pathway may include the recently identified protein, retinoic acid inducible gene-1 (11). After the production of IFN-, the transcription of several IFN-stimulated genes (ISGs) is up-regulated through the IFN signaling cascade. However, accumulating evidence indicates that some ISGs may be induced by IRF3 in the absence of IFN production, providing an IFN-independent pathway for activating certain ISGs (12).
In the IFN signaling cascade, the STAT1 pathway is critical for regulating the expression of most ISGs. Mice lacking an intact STAT1 gene are defective in their ability to mount many classical responses to IFN- and IFN- and are highly susceptible to numerous viral infections (13, 14, 15, 16). However, STAT1-deficient 129 (STAT129) mice have been reported to be more resistant than IFN-R- and IFN-R-deficient mice to infection with Sindbis virus (SV) or murine CMV (MCMV) (17), indicating that some antiviral responses may be mediated by STAT1-independent mechanisms (18, 19, 20). Certain viruses may compromise STAT1 functions (21, 22, 23), requiring the STAT1-independent pathway to provide the host with an alternative mechanism for IFN-dependent signaling. Indeed, the DEN nonstructural protein NS4B has recently been demonstrated to inhibit IFN signaling by blocking STAT1 activity in vitro (24), suggesting that the IFN system may need to mediate its responses against DEN infection in vivo at least partially through the STAT1-independent pathway. Therefore, to determine whether IFNs control DEN infection in vivo via the STAT1-dependent or STAT1-independent pathway, we examined the role of STAT1 in resolving primary DEN infection in mice.
Using two different DEN serotypes and STAT1–/– 129 mice carrying a deletion mutation in the DNA binding domain of STAT1 (13) or the N-terminal domain of STAT1 (14), we demonstrate in this study that the IFN-mediated responses to primary DEN infection involve both the STAT1-dependent and STAT1-independent pathways. Virologic analyses revealed that the STAT1 pathway controls the initial viral burden, whereas the STAT1-independent response ultimately resolves the infection in mice. Immunologic analyses showed that the STAT1-independent pathway involves some of the major immunoregulatory effects of IFNs, including 1) early activation of B and NK cells and 2) up-regulation of MHC class I molecules on macrophages and dendritic cells (DCs). Infection of bone marrow-derived DC cultures with either DEN or SV, another positive-strand RNA virus, confirmed the role of STAT1 in mediating early viral clearance. Additionally, these studies demonstrated that both DEN and SV can induce IFN- production in DC cultures via the STAT1-independent pathway, and that STAT1 normally inhibits IFN- production by these cells. These findings provide new insights on IFN-dependent, STAT1-dependent vs IFN-dependent, STAT1-independent responses during viral infections in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Mice were bred and maintained under specific pathogen-free conditions in the animal facility at University of California-Berkeley. 129 Sv/Ev mice lacking receptors for both IFN- and IFN- (AG129) and for IFN- alone (IFN-R–/– 129/Sv/Ev (A129)) were obtained from Dr. S. Virgin (Washington University School of Medicine, St. Louis, MO). Wild-type (WT129) and STAT1–/– 129/Sv/Ev mice carrying a deletion of the N-terminal domain of STAT1 (STAT129) were purchased from Taconic Farms. 129/Sv/Ev mice bearing a deletion in the DNA binding domain of the STAT1 gene were obtained from Dr. J. Durbin (Ohio State University, Columbus, OH). DEN-infected mice were kept in the biosafety level 2 suites of the animal facility, and all procedures involving mice were approved by the animal care and use committee at University of California-Berkeley.
Viruses
DEN2 PL046 (Taiwanese isolate, Ae. albopictus C6/36 mosquito cell-adapted) and DEN1 Mochizuki (mouse brain-adapted) strains were originally obtained from Drs. H.-Y. Lei (National Cheng Kung University, Tainan, Taiwan) and R. Tesh (University of Texas Medical Branch, Galveston, TX), respectively. Viral stocks were amplified in C6/36 cells, titrated by plaque assays using BHK-21 cells, and concentrated via ultracentrifugation as previously described (6). Mice at 5–6 wk of age were infected i.v. with 107 or 108 PFU of DEN2 or 4.4 x 104 PFU of DEN1 in 250 µl. For infection of bone marrow-derived DCs with SV, a plasmid encoding the full-length Sindbis genome plus GFP (25) was obtained from Dr. I. Frolov (University of Texas Medical Branch, Galveston, TX). The plasmid was purified using the Qiagen Plasmid Midi kit and then linearized with XhoI for in vitro transcription. RNA was transcribed in the presence of m7G cap analog using RiboMAX Large Scale RNA Production System-SP6 (Promega) according to the manufacturer’s instructions. The transcription reaction mixture was electroporated into BHK-21 cells, and virus was harvested 24 h after electroporation, as previously described (25). Virus was titrated by standard plaque assays using BHK-21 cells, with fixing and visualization of plaques on day 2 postinfection (p.i.).
Quantitation of virus in infected mice
Mice were euthanized by isofluorane inhalation just before obtaining whole blood via cardiac puncture. Serum and other tissue samples were harvested, processed, and tested for the presence of infectious virus by both direct and amplified plaque assays, as previously reported (6).
Flow cytometric analysis of spleen cells from infected mice
After harvest, spleens were disrupted between the frosted ends of two glass slides in complete RPMI 1640 medium, which consists of RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS (HyClone), 10 mM HEPES, 200 mM L-glutamine, 10,000 U/ml penicillin and streptomycin, 50 mM 2-ME, 1% nonessential amino acids, and 1% sodium pyruvate. Single-cell suspensions were prepared in FACS buffer (PBS/5% FCS/10 mM HEPES/5 mM EDTA/0.05% NaN3), blocked with anti-mouse CD16 (clone 24G2) hybridoma supernatant, and stained with directly conjugated mAbs against mouse cell surface markers as previously reported (6). Combinations of the following mAbs labeled with FITC, PE, or PE-Cy5 were used: anti-CD3, anti-H-2Kb, anti-I-A/I-Eb, and anti-DX5 (BD Pharmingen); anti-CD19, anti-CD69, and anti-CD11c (eBioscience); and anti-F4/80 (Caltag Laboratories). For intracellular staining of IFN-, anti-IFN--PE (Caltag Laboratories) and the Fixation and Permeabilization kit (eBioscience) were used, and all procedures were performed according to the manufacturer’s instructions. An EPICS XL-MCL (Beckman Coulter) flow cytometer was used to acquire at least 100,000 events/sample. Data were reanalyzed using FlowJo software (TreeStar).
Bone marrow-derived DCs
To generate CD11c+ DCs from bone marrow, the femur and tibiae were isolated and processed based on a published protocol (26). Briefly, the marrow was flushed out with complete RPMI 1640 medium injected with a syringe. After lysis of RBC in a buffered ammonium chloride solution, bone marrow cells were washed and then plated at 2 x 106 cells/ml in complete RPMI 1640 and 50 ng/ml murine Flt3 ligand (Flt3L; Research Diagnostics). Culture medium was removed and replaced with fresh medium containing Flt3L every 3 days. On day 12, cells were harvested using HBSS/5 mM EDTA solution, washed with complete RPMI 1640, and processed for flow cytometric analysis and infection with DEN2 or SV. For flow cytometry, cells were incubated with anti-CD16 to block FcRs, stained with anti-CD11c-PE-Cy5, anti-B220-PE, and anti-Ly6C-FITC, and analyzed on a Beckman Coulter flow cytometer, as described above. For infection with DEN2 PL046 strain or SV, 106 cells/well in a 12-well plate were infected at a multiplication of infection (MOI) of 10 in a total volume of 0.5 ml. After 2-h incubation in a humidified 37°C/5% CO2 incubator, cells were extensively washed with PBS and then cultured in complete RPMI 1640 for 6 days. Culture supernatants were harvested daily, stored frozen at –80°C, and used later to test for the presence of infectious virus and IFNs. Standard plaque assays and ELISAs were performed to quantitate the levels of infectious virus and IFNs, respectively, in the culture supernatant. The mouse IFN- ELISA Ready-SET-Go (eBioscience) and mouse IFN- ELISA (PBL Biomedical Laboratories) kits were used as recommended by the manufacturer. The mouse IFN- ELISA kit detects murine IFN-A, -1, -4, -5, -6, and -9. The OD at 450 nm of each sample was measured using an Elx808 microplate reader (Bio-Tek Instruments) and KCjunior software (Bio-Tek Instruments). Cytokine levels were expressed as the number of picograms per milliliter. The limits of detection of IFN- and IFN- were 15 and 10 pg/ml, respectively.
Statistical analyses
Kaplan-Meier survival curves were generated using PRISM software, version 3.0cx (GraphPad). The same software was used to assess statistical significance by Student’s t test and one-way ANOVA with Tukey’s multiple comparison test. Comparisons were considered significant at p < 0.05.
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Results |
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Using the doubly deficient AG129 mice, we have demonstrated that the combined activities of the two IFNRs are essential for resistance to DEN-induced disease (7). Because both IFNRs signal through STAT1, the present study examined the contribution of STAT1 to controlling primary DEN infection in mice. STAT129 and WT129 mice were infected i.v. with 4.4 x 104 PFU of the DEN1 Mochizuki strain or 108 PFU of the DEN2 PL046 strain, and the development of DEN-induced paralysis was monitored daily until day 30 p.i. After infection with a mouse-adapted strain of DEN1, 28% of STAT129 and 7% of WT129 mice exhibited hind leg paralysis (Fig. 1A). Similarly, 17% of STAT129 and 7% of WT129 mice developed disease after infection with a DEN2 strain that has not been mouse-adapted (Fig. 1B). In contrast, 100% of AG129 mice infected with either DEN1 or DEN2 showed paralysis. As expected, infectious virus was detected in the brain and spinal cord of the paralytic mice by standard plaque assays (data not shown). At 107 PFU of DEN2, 0% of STAT129 mice, but almost 80% of AG129 animals, succumbed to DEN-induced paralysis (data not shown). These results indicate that STAT1 plays a minimal role in protection against DEN-induced disease in mice. To confirm these findings, STAT1–/– mice carrying a different mutant stat1 allele were tested. As expected, STAT1–/– 129 mice lacking a portion of the DNA binding domain of the STAT1 gene (DBD KO) were resistant to disease after infection with either DEN1 (Fig. 1C) or DEN2 (Fig. 1D). Therefore, we conclude that the IFN system protects against DEN disease in 129 mice via a STAT1-independent pathway.
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We have previously shown that 129 mice lacking only the IFN-R (A129) are unable to control DEN2 titers at early times p.i., but are able to resist DEN-induced disease (7). Therefore, we questioned whether STAT1 would have a similar role in mediating early viral clearance. Amplified plaque assays (7), which represent a sensitive measure for detecting infectious virus, revealed that STAT129, A129, and AG129, but not WT129, mice harbored infectious DEN2 on day 1 p.i. in several tissues (Table I). By day 3 p.i., STAT129 mice had undetectable amounts of virus in the serum, liver, and spleen, whereas both A129 and AG129 mice still carried infectious virus in these tissues. By day 7 p.i., STAT129 and A129 mice contained undetectable levels of virus in all tissues examined, whereas AG129 mice had virus in the brain and spinal cord. Additionally, in our previous study, G129 mice lacking IFN-R alone were shown to have no infectious DEN at these early time points p.i (7). Considered together, these findings demonstrate a role for STAT1 in limiting early viral load, and comparison of the phenotype between STAT129 and A129 mice suggests that the STAT1 activity responds to the IFN-R engagement. Therefore, the data reported above reveal the contributions of both STAT1-dependent and -independent systems to the control of primary DEN infection in mice.
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Consistent with our results, STAT129 mice have been shown to be more resistant than AG129 mice to infection with SV or MCMV (17). However, little is known about the nature of this STAT1-independent signaling in IFN-induced biological responses in vivo. Therefore, we sought to determine which of the major IFN-mediated responses might be regulated by the STAT1-independent mechanism in mice with primary DEN infection. WT A/J, 129, and C57BL/6J mice with primary DEN infection have an increased number of activated B and NK cells in the spleen on day 3 p.i. (6) (S. Shresta and E. Harris, unpublished observations), raising the possibility that IFNs induce certain B and NK cell-mediated responses early during primary DEN infection in mice. In 129 mice, this IFN action may be STAT1 independent, because infectious virus is no longer detectable in the spleen of STAT129 mice on day 3 p.i., as shown in Table I. Therefore, we tested whether the early activation of B and NK cells depends on the IFN-mediated, STAT1-independent pathway by performing flow cytometric analysis of spleen cells from WT129, STAT129, and AG129 mice.
The activation status of CD19+ B cells and CD3–DX5+ NK cells was assessed by their expression of CD69, an early activation marker, and activated NK cells were further phenotyped for intracellular IFN- expression. As expected, uninfected WT129, STAT129, and AG129 mice had similar numbers of spleen cells (Fig. 2A), and they contained a low percentage of CD19+CD69+ (activated B), CD3–DX5+CD69+ (activated NK), and CD3–DX5+IFN-+ (activated NK) splenocytes (Fig. 2, B–D). Compared with uninfected mice, the number of lymphocytes in spleens from all three types of mice increased after 3 days of infection with DEN2, with approximately twice as many splenocytes in AG129 mice as in WT129 or STAT129 mice. However, both WT129 and STAT129 spleens had higher percentages and numbers of CD69+CD19+ activated B cells and CD3–DX5+ NK cells expressing either CD69 or IFN- than spleen cells from AG129 mice (Table II and Fig. 2). This activation of immune cells was not due to LPS contamination, because only live, not UV- or heat-inactivated, DEN preparations induced significant increases in the number of activated B and NK cells (data not shown). These results indicate that the early activation of B and NK cells in 129 mice with primary DEN infection is induced by the IFN-dependent, STAT1-independent response in the absence of STAT1. To confirm the role of the STAT1-independent pathway in mediating NK and B cell responses on day 3 p.i., the activation status of NK and B cells was examined at a time point earlier than day 3. On day 1 p.i., the total numbers of activated NK and B cells in both WT129 and STAT129 mice were lower than the day 3 p.i. values (data not shown), confirming that DEN-infected mice have higher numbers and percentages of activated NK and B cells on day 3 p.i. than on day 1 p.i.
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Besides regulating the activities of B and NK cells, IFNs modulate the function of other immune cells, including professional APCs. One of the shared functions of IFN- and IFN- is the up-regulation of MHC class I molecules on APCs. Therefore, WT129, STAT129, and AG129 mice were infected with DEN2, and spleen cells on day 3 p.i were analyzed for the expression of MHC class I molecules by flow cytometry. In uninfected mice, F4/80+ macrophages expressed basal levels of H-2Kb (Fig. 3A). After 3 days of infection, F4/80+ cells in spleens of both WT129 and STAT129 mice had higher levels of H-2Kb expression than uninfected mice, whereas this difference was not observed between uninfected and infected AG129 mice (Fig. 3, A and B). Similarly, CD11c+ DCs from WT129 and STAT129, but not AG129, mice displayed an up-regulation of H-2Kb (Fig. 3, C and D). Therefore, in the absence of STAT1, IFNs use a STAT1-independent pathway to increase MHC class I levels on the surface of macrophages and DCs in 129 mice with primary DEN infection.
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Based on the finding that the IFN system regulates MHC class I levels on DCs from DEN-infected mice, we next focused our studies on DCs as a possible source of IFN production during DEN infection. DCs may be the key APCs and initial targets of DEN in vivo (27, 28, 29, 30, 31), although the exact phenotype of the cell types that support DEN replication in vivo is presently unclear. Because small numbers of virus-infected cells in mice at early time points after DEN infection are difficult to quantitate (S. Shresta and E. Harris, unpublished observations), we generated bone marrow-derived DC cultures and examined whether IFNs and STAT1 controlled IFN production as well as viral infection in DEN-infected cultures. Bone marrow cells from WT129, STAT129, and AG129 mice were cultured in the presence of Flt3L for 12 days, and the resulting population of cells was phenotyped by flow cytometric analysis. Approximately 90% of cells from each mouse genotype were CD11c+ DCs, and the remaining contaminants were Gr1+ granulocytes and F4/80+ macrophages, but not B, T, or NK cells, as indicated by the absence of staining for CD19, CD3, and DX5, respectively. Additionally, <2% of the CD11c+ DC population included plasmacytoid DCs that coexpressed B220 and Ly6C.
To test the susceptibility of these bone marrow-derived DCs to DEN infection, cells were incubated with DEN2 at an MOI of 10 for 2 h, washed to remove unbound virions, and analyzed for infectious virus in the culture supernatant at various times p.i. by standard plaque assay. At all time points after infection, infectious virus was undetectable in cultures from WT129 mice, whereas high levels of infectious DEN were present in AG129 cultures throughout the experimental period (Fig. 4A). In STAT129 cultures, viral titers were almost as high as AG129 levels on days 1 and 2 p.i., but they gradually decreased starting on day 3 p.i to an undetectable level by day 6 p.i. Microscopically, no cytopathic effects were observed at any time point p.i. in either wild-type or STAT1-deficient cultures, whereas AG129 cultures exhibited increasing levels of cellular cytotoxicity (cell rounding and detachment) starting on day 2 p.i. By day 6 p.i., a vast majority of AG129 cells were dead; in contrast, both WT129 and STAT129 cultures were confluent with live cells. Taken together, these results demonstrate that IFNs are essential for limiting viral growth in bone marrow-derived DC cultures in vitro at all time points p.i., and that the STAT1 pathway controls early viral replication, whereas a STAT1-independent pathway reduces later viral titers. These findings support the in vivo data shown in Table I, validating the use of this in vitro model of DEN infection for additional studies.
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and IFN- production by these cells. Fig. 4B shows IFN- levels, as measured by an ELISA that specifically detects murine IFN-A, -1, -4, -5, -6, and -9. At all time points p.i., low amounts of IFN- were detected in WT129 culture supernatants, and the levels of IFN- in AG129 cultures were even lower than those in WT129 cultures. Similar to AG129 results, on day 1 p.i., the level of IFN- in STAT129 culture supernatants was lower than that in WT129 cultures; however, on days 2, 3, and 4 p.i., IFN- levels in STAT129 culture supernatants were dramatically higher than those in WT129 cultures. These results reveal that STAT1-deficient cells produce IFN- at later times after infection with DEN.
Compared with IFN- results, IFN- levels in the same DEN-infected DC cultures were low, as measured by murine IFN--specific ELISA (Fig. 4C). Both WT129 and AG129 DC culture supernatants contained IFN- levels that were low at all time points tested, whereas STAT129 culture supernatants had significantly higher amounts of IFN- than WT129 and AG129 cultures starting on day 2 p.i. In STAT129 cultures, the level of IFN- was highest on day 2 p.i. and then progressively decreased at later time points. These results show that STAT1-deficient cells also produce IFN- in response to DEN infection at later time points p.i. Together, the results presented in Fig. 4, B and C, confirm that STAT1-deficient cells use the IFN-dependent, STAT1-independent pathway to control DEN infection at later times p.i. In addition, sera from DEN2-infected STAT129 mice contained IFN- on day 1 p.i. (1615.3 ± 95.4 pg/ml) and day 3 p.i. (60.0 ± 121.8 pg/ml) as well as IFN- on day 1 p.i. (759.5 ± 215.1 pg/ml) and day 3 p.i. (71.4 ± 40.8 pg/ml), and both IFNs were undetectable by day 7 p.i. Therefore, both in vivo and in vitro findings support the conclusion that the STAT1-independent pathway involves both IFN- and IFN- production.
STAT1-dependent and STAT1-independent responses control SV infection in bone marrow-derived DC cultures from 129 mice
To extend the results described above to another viral infection model, parallel experiments were performed using bone marrow-derived DC cultures infected with SV, a member of the family Togaviridae and the genus Alphavirus. Similar to DEN, SV is a ssRNA virus, and STAT129 mice are less susceptible than AG129 mice to infection with SV (17). Fig. 5A shows infectious SV titers in WT129, STAT129, and AG129 DC culture supernatants at various time points p.i., as determined by plaque assay. Similar to results obtained from DEN-infected cultures (Fig. 4A), the level of infectious virus was consistently higher in AG129 culture supernatants compared with WT129 and STAT129 cells, confirming an essential role of the IFN system in controlling SV replication. Unlike DEN, SV established productive infection in WT129 DC cultures. In both WT129 and STAT129 culture supernatants, viral titers peaked on day 1 p.i. and progressively decreased at later time points. The levels of infectious SV in STAT129 cultures tended to be higher than those in WT129, but lower than AG129 values, at all time points p.i. By day 6 p.i., a low level of SV was still present in STAT129 cultures, whereas infectious virus was no longer detectable in WT129 cultures, which consisted of normal, viable cells. Similar to findings obtained from DEN infection studies, these results demonstrate that the STAT1-independent pathway controls viral titers at later times p.i.
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and IFN- levels in SV-infected DC culture supernatants by ELISA. Significant amounts of IFN- were observed in both WT129 and STAT129 cultures, whereas levels of IFN- were just above background in AG129 cultures (Fig. 5B). IFN- levels in both WT129 and STAT129 cultures were similar, and they decreased over time, reflecting the drop in viral titers with time (Fig. 5A). In comparison with IFN- induction, at all time points p.i., little or no IFN- was present in the WT129 and AG129 culture supernatants, but significant amounts of IFN- were detected in STAT129 cultures (Fig. 5C). Fig. 5, B and C, shows that STAT1-deficient cells produce IFN- and IFN- in response to SV infection, supporting the conclusion that the STAT1-independent pathway in SV-infected DC cultures is also IFN dependent. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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A previous study using MCMV- or SV-infected STAT1–/– 129 mice was the first to document the importance of the STAT1-independent pathway in mediating protective responses against viral infections in vivo (17). The present study extends the published observation in several directions. First, we have demonstrated the role of STAT1-dependent and STAT1-independent responses, respectively, as initial and subsequent pathways for resolving a viral infection in vivo. Second, we have identified specific IFN-dependent, STAT1-independent immune responses in vivo. Finally, using DEN- or SV-infected bone marrow cultures in vitro, we have confirmed that the STAT1-independent pathway is IFN dependent.
The early vs late roles of the STAT1-dependent and STAT1-independent pathways in clearing viral infection were defined based on kinetic experiments involving DEN infection in vivo and in vitro as well as SV infection in vitro. The STAT1 pathway limits infectious viral titers on day 1 p.i., whereas the STAT1-independent pathway decreases viral burden by day 3 p.i. On both days 1 and 3 p.i., IFN-R activities control DEN replication, and our previous study with mice lacking IFN-R alone revealed that the IFN-R pathway does not participate in early DEN clearance (7). Based on these findings, the STAT1 pathway involves IFN-, but not IFN-, receptor-mediated responses; however, the STAT1-independent pathway mediates both IFN-R and IFN-R activities. In support of this conclusion, studies using SV-infected A129 mice have demonstrated that IFN- receptors alone play a critical role in controlling viral replication during the early stages of infection before a specific Ab response develops (32, 33). Furthermore, studies with SV-infected DC cultures have indicated that the IFN-R activity uses the dsRNA-dependent protein kinase (PKR) pathway at early time points p.i., whereas IFN-Rs act independently of PKR at later time points p.i. (34). Moreover, in STAT1–/– bone marrow-derived macrophages, the IFN--dependent, STAT1-independent signaling does not require PKR (17, 20). Taken together, these observations suggest that in vivo, the early STAT1 pathway may be mediated by PKR, whereas the late STAT1-independent pathway is likely to be PKR independent. A recent study has demonstrated that STAT3 is activated by IFN- in STAT1-deficient mouse embryo fibroblasts, leading to the transcription of several genes that are normally induced by STAT1 in WT cells (35). Therefore, STAT3 is a plausible candidate for transducing some of the STAT1-independent responses in vivo. Future experiments using 129 mice with primary DEN infection should help determine the roles of both PKR-independent and STAT3-dependent mechanisms in mediating the STAT1-independent pathway in vivo.
Besides identifying the kinetic difference of the STAT1-dependent and STAT1-independent pathways, this study defines specific IFN-dependent, STAT1-independent immune responses in vivo. Specifically, in STAT129 mice with primary DEN infection, the STAT1-independent pathway includes the early activation of splenic B cells. At present, the significance of the early activation of B cells in mice with primary DEN infection is unclear, because B cell-deficient mice resolve primary DEN infection as efficiently as WT mice (7). Similar to DEN, rotavirus, a dsRNA virus of the family Reoviridae, also induces a massive increase in the number of activated B cells at early times p.i. (36, 37), and B cell-deficient mice clear primary rotavirus infection (38). However, B cell-deficient mice are not protected against rechallenge with rotavirus, indicating a requirement for B cells in immunity against rotavirus reinfection (38). Similarly, B cells may play a more important role during secondary DEN infection in vivo. According to the Ab-dependent enhancement theory, DEN serotype-cross-reactive Abs at subneutralizing concentrations enhance viral infection of FcR-positive cells, such as macrophages, during sequential DEN infection (39). In 129 mice, the STAT1-dependent and STAT1-independent pathways may shape the memory B cell repertoire during primary DEN infection as well as directly regulate B cell functions during secondary DEN infection.
In addition to the early activation of B cells, another STAT1-independent response in STAT129 mice with primary DEN infection is the early activation of NK cells. Again, the role of NK cells in protection against primary DEN infection is presently unclear, because 129 mice treated with anti-asialo-GM1 Abs, which deplete NK cells in vivo, have a normal ability to clear primary DEN infection (S. Shresta and E. Harris, unpublished observations). However, experiments using better loss-of-function models for murine NK cells may show a role for NK cells in limiting primary and/or secondary DEN infection in mice. In comparison, NK cells are essential for early control of MCMV replication in mice (40, 41, 42), suggesting that in STAT1-deficient mice, the STAT1-independent pathway may be clearing MCMV infection by regulating NK cell functions. NK cells use both effector functions, namely, cell-mediated cytotoxicity and IFN- production, to limit MCMV infection in mice (43). Similar to results obtained from 129 mice with primary DEN infection, a large number of splenic NK cells in MCMV-infected mice express intracellular IFN- at early time points p.i. (44). Furthermore, the observation that in 129 mice with primary DEN infection, spleens from STAT1-deficient mice have higher numbers of IFN--expressing NK cells than WT spleens agrees with a published finding that the STAT1 response inhibits IFN- production by NK cells in mice infected with lymphocytic choriomeningitis virus, a negative-stranded RNA virus of the family Arenaviridae (45). Therefore, during viral infections in general, the STAT1 pathway may negatively regulate early IFN- production by NK cells to avoid immunopathology, whereas the STAT1-independent pathway may activate NK cells to control rising viral titers at later times p.i.
Besides NK and B cell activation, the STAT1-independent response induces the up-regulation of MHC class I molecules on professional APCs in mice with primary DEN infection. Several reports have demonstrated that in vitro infection of mammalian cells by a variety of flaviviruses, including DEN, up-regulates cell surface expression of MHC class I molecules (46, 47, 48). Although this phenomenon appears to be a by-product of flavivirus assembly, it may allow flaviviruses to evade NK cell attack (49). However, based on the observation that flavivirus-infected cells are highly susceptible to CTL-mediated lysis in vitro, King et al. (48) have hypothesized that flaviviruses, as part of their survival strategy in vivo, may generate massive numbers of low affinity T cells that clear virus poorly. Therefore, the up-regulation of MHC class I molecules on macrophages and DCs may have important implications in vivo, because a major role of MHC molecules is to present viral Ags to specific T cells, and both macrophages and DCs may be crucial targets of DEN infection in vivo.
Bone marrow-derived DC cultures from both STAT129 and AG129, but not WT129, mice are highly susceptible to DEN infection at early times p.i. At later time points p.i., the IFN-dependent, STAT1-independent response leads to a clearing of infectious DEN in DC cultures from STAT129 mice, confirming our in vivo data. This in vitro model of DEN infection using nontransformed, primary cells that are more relevant to human infection should allow us to address the IFN mechanism of action at the cellular level. However, the cellular sources of IFNs in these DEN-infected DC cultures have yet to be identified. Both the conventional DCs as well as the small number of contaminating plasmacytoid DCs and non-DCs may be contributing to the production of IFNs in these bone marrow-derived DC cultures. Future studies using our mouse model of primary DEN infection should uncover the exact mechanisms by which the IFN system mediates antiviral responses.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by the National Institutes of Health (National Research Scientist Award 1F32AI51070-01 to S.S.) and the Ellison Medical Foundation (Grant 1D-1A-0031 to E.H.). 
2 Address correspondence and reprint requests to Dr. Eva Harris, Division of Infectious Diseases, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720-7360. E-mail address: eharris{at}berkeley.edu
3 Abbreviations used in this paper: DEN, dengue virus; DC, dendritic cell; DEN1, DEN serotype 1; DEN2, DEN serotype 2; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; Flt3L, Flt3 ligand; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; MCMV, murine CMV; MOI, multiplication of infection; p.i., postinfection; PKR, dsRNA-dependent protein kinase; SV, Sindbis virus; WT, wild type.
4 Current address: La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
Received for publication February 1, 2005. Accepted for publication June 30, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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. Proc. Natl. Acad. Sci. USA 98:6674.-6679. 
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