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The Journal of Immunology, 2006, 177: 6301-6307.
Copyright © 2006 by The American Association of Immunologists, Inc.

TLR3 Deletion Limits Mortality and Disease Severity due to Phlebovirus Infection1

Brian B. Gowen2,*, Justin D. Hoopes{dagger}, Min-Hui Wong*, Kie-Hoon Jung*, Kevin C. Isakson*, Lena Alexopoulou{ddagger}, Richard A. Flavell§ and Robert W. Sidwell*

* Institute for Antiviral Research, Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT 84322; {dagger} Department of Biology, Utah State University, Logan, UT 84322; {ddagger} Centre d’Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique-Institut National de la Sante et de la Recherche Medicale, Marseille, France; and § Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3–/– mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3–/– mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-{gamma}, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3–/– mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system has the inherent capacity to sense a wide spectrum of infectious agents through a series of germline-encoded proteins known as TLRs (1). In most cases, development of protective immunity against invading pathogens requires timely recognition through interactions with one or more members of the TLR family of proteins. Signaling pathways triggered through these receptors serve to bridge the innate and adaptive components of immunity through the maturation of macrophages and dendritic cells and the induction of proinflammatory cytokines that promote robust interaction between these specialized APCs and T cells (2). TLR3 was first described as a cellular receptor for dsRNA (3), a molecular pattern associated with viral infection. Initially, in vivo evidence demonstrating the involvement of TLR3 in host defense against viral pathogens was lacking (4); however, more recent investigations indicate that TLR3 contributes significantly to the antiviral immune response (5, 6, 7, 8). Animal models were used in several of these studies, providing compelling evidence that TLR3 plays a significant role in vivo. Further evidence suggesting that TLR3 plays a vital role in antiviral immunity is found in several recent reports that have demonstrated viral evasion strategies targeting TLR3 signaling (9, 10, 11, 12) because the process of viral evolution most likely targets components that serve essential functions in protecting the host.

Several viruses of the Bunyaviridae family, genus Phlebovirus, can cause considerable disease in humans and ruminants. Historically, Rift Valley fever virus (RVFV)3 and Sandfly fever virus have accounted for much of the disease. Sandfly fever was a major problem in World War II in the Mediterranean area in which ~19,000 allied troops were afflicted, with most requiring hospitalization (13). RVFV is the only phlebovirus that can lead to fatal disease in humans (14), and has caused several severe epidemics throughout Africa and the Middle East (13). It has received select agent status from the Department of Health and Human Services and the U.S. Department of Agriculture, and has been designated a category A priority pathogen by the National Institute of Allergy and Infectious Diseases, underscoring its potential impact on global agriculture and public health in the event of deliberate release. The stringent requirements for working with pathogenic strains of RVFV make it inaccessible to most researchers. Closely related to RVFV, Punta Toro virus (PTV) is a less pathogenic phlebovirus, endemic throughout rural Panama (15). Pifat and Smith (16) initially reported work describing PTV infection of weanling mice that produces a fatal hepatic disease similar to that caused by RVFV in humans and livestock. This animal model of phlebovirus infection has facilitated numerous investigations of promising antivirals, and has served as a highly predictive substitute for modeling Rift Valley fever and sandfly fever phleboviral disease (17, 18, 19, 20, 21, 22).

PTV is an ssRNA virus that generates dsRNA intermediates as it replicates in host cells. Previous research characterizing the PTV infection model in C57BL/6 mice demonstrated that survival outcome is age dependent (16). Infection in mice of 3–4 wk of age produced lethality in ~90% of animals. As mice matured and reached 7–8 wk of age, the ability to clear the virus and survive infectious challenge developed (16). We have discovered recently that added stress associated with daily handling and observation results in increased susceptibility of older mice, with fatality rates ranging from 60 to 90% (B. Gowen, unpublished observations). Thus, to investigate the potential involvement of TLR3 in protective immunity, we challenged 8-wk-old TLR3-deficient and wild-type mice with PTV and evaluated survival outcome, viral burden, liver disease, and cytokine and chemokine responses during the course of infection. Resulting cytokine profiles lead to further evaluation of the contribution of IL-6 to pathogenesis and host defense in the PTV infection model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

TLR3–/– mice were derived at Yale University by R. Flavell (New Haven, CT) (3) and backcrossed extensively onto a C57BL/6 background. A breeding colony was established and housed in the animal facility at Utah State University under specific pathogen-free conditions. C57BL/6 mice (wild-type) and IL-6-deficient mice were obtained from The Jackson Laboratory. Unless otherwise specified, carefully age-matched 8-wk-old female mice were used. All animal procedures used in these studies complied with guidelines set by the U.S. Department of Agriculture and Utah State University Animal Care and Use Committee.

Virus

PTV, Adames strain, was provided by D. Pifat (U.S. Army Medical Research Institute for Infectious Diseases, Ft. Detrick, Frederick, MD). Virus stocks were prepared following four passages of the original virus stock through LLC-MK2 cells (American Type Culture Collection).

Mouse PTV infection studies

For survival studies, groups of 10–20 TLR3–/–, IL-6–/–, or wild-type mice were inoculated by s.c. injection with 5 x 103 PFUs of PTV. The mice were observed for weight loss and mortality for 14 days. Groups of three to four animals were used for studies comparing temporal systemic and liver virus burden, cytokine and chemokine levels, and liver disease in TLR3–/– and wild-type mice. Mice were sacrificed at the indicated times throughout the course of infection, and livers were scored on a scale of 0–4 for hepatic icterus, 0 being normal and 4 being maximal yellow discoloration. Serum was collected for assaying alanine aminotransferase (ALT) activity, and virus titers were determined for both liver and serum samples, as described below.

Liver and serum virus titers

Virus titers were assayed using an infectious cell culture assay, as previously described (19). Briefly, a specific volume of liver homogenate or serum was serially diluted and added to triplicate wells of LLC-MK2 cells in 96-well microplates. The viral cytopathic effect was determined 5–6 days postvirus exposure, and the 50% end points were calculated, as described (23).

ALT determinations

Detection of elevated ALT activity in serum serves as an indicator of liver dysfunction associated with infection of hepatotropic viruses such as PTV. Serum ALT levels were measured using the ALT (serum glutamic pyruvate transaminase) reagent set purchased from Pointe Scientific, following the manufacturer’s recommendations. The reagent volumes were adjusted for analysis on 96-well microplates.

Cytokine and chemokine multiplex profiling

Liver and systemic expression of 16 cytokines (IL-1{alpha}, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, MCP-1, IFN-{gamma}, TNF-{alpha}, MIP-1{alpha}, GM-CSF, and RANTES) was evaluated using Q-Plex mouse cytokine arrays (BioLegend), as recommended by the manufacturer. Liver homogenates in MEM (0.1 g/ml) and serum were stored at –80°C until time of analysis.

Statistical analysis

Log-rank analysis was used to evaluate differences in survival using JMP statistical software (SAS Institute). Student’s t test (two tailed) was performed to analyze differences in virus titers, serum ALT, and cytokine levels. Wilcoxon ranked sum analysis was used for mean liver score comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Increased resistance of TLR3–/– mice to PTV infection

To investigate whether TLR3 contributes significantly to in vivo antiviral defense against phleboviral infection, 8-wk-old TLR3–/– and wild-type mice were challenged with 5 x 103 PFU of PTV and observed for 14 days. Interestingly, TLR3–/– mice were significantly more resistant to an LD90 challenge dose of PTV than wild-type animals (Fig. 1). Heightened resistance does not appear to be gender specific, as similar results were obtained with male mice (data not shown). We next evaluated systemic and liver viral loads, as well as liver damage and discoloration associated with PTV-induced disease in groups of mice sacrificed at various intervals following infection. To lessen disease severity and limit mortality so as to facilitate sampling past day 3 of infection, handling stress was limited to a single weight determination the day before challenge. Consequently, only 3 (one per group) of the combined 10 total animals in the days 4–6 wild-type sacrifice groups and none of the TLR3–/– mice died before sacrifice. Despite having similar peak serum virus titers, levels in TLR3–/– mice began to decrease by day 3, whereas high levels persisted in the wild-type animals out to 4 days (Fig. 2A). As shown in Fig. 2B, no appreciable difference in liver virus burden was observed between wild-type and TLR3–/– mice. However, liver disease was significantly limited in TLR3–/– mice, as indicated by reduced serum levels of ALT (Fig. 2C). Although not as dramatic, further evidence of reduced liver damage in TLR3–/– mice was seen by visual inspection of hepatic icterus in infected mice on day 4 of infection (Fig. 2D). Due to mortality and severe disease before time of sacrifice, late stage serum (and in one case liver) samples for several wild-type mice were unobtainable or insufficient for analysis. Thus, the magnitude of infection and disease reported for wild-type mice after day 3 is most likely underestimated because we were unable to include some of the sickest animals in the analysis.


Figure 1
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  		FIGURE 1. TLR3–/– mice demonstrate increased resistance to PTV infection. TLR3–/– and wild-type mice (20/group) were challenged with PTV and observed daily for mortality for a duration of 14 days. Survival curves shown are representative of three similar experiments. *, p = 0.002 compared with wild-type mice.

 

Figure 2
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  		FIGURE 2. TLR3–/– mice have reduced liver disease during late stages of infection. Analysis of serum (A) and liver virus titers (B), ALT release (C), and hepatic discoloration (D) (increasing in severity from 0 to 4) at indicated sacrifice times following infection with PTV. The limits of detection for the virus titer assays were 2.8 log10 50% cell culture infectious doses (CCID50) per g of liver tissue and 1.8 log10 CCID50 per ml of serum. ALT activity present in serum serves as an indirect measure of liver injury. Values represent the means and SDs of groups of three to four animals. Due to mortality or poor condition of some of the wild-type mice before time of sacrifice at late stages of infection, little to no serum could be obtained for several mice. Thus, ALT and systemic virus data for day 4 were from three mice, and days 5 and 6 were from two mice per group. Profiles consistent with liver disease shown here were also observed in a similar experiment. *, p < 0.05; **, p < 0.01 compared with wild-type mice by Student’s t test.

 
Differences in liver cytokine levels between wild-type and TLR3–/– mice during PTV infection

Viral replication does not appear to be responsible for the observed liver damage in wild-type animals as hepatic viral loads were comparable to those seen in TLR3–/– mice. Thus, we examined liver cytokine profiles following PTV infection to assess potential immune-mediated causes of liver dysfunction associated with increased mortality in wild-type mice. With the exception of a single TLR3–/– mouse, IL-6 and IFN-{gamma} levels were collectively higher in wild-type mice on day 3 of infection (Fig. 3, A and B). By day 4 postvirus challenge, which coincides with the time at which mice begin to die from infection, average IL-6 and IFN-{gamma} levels in wild-type mice were 6.6- and 3.6-fold greater, respectively, than those observed for TLR3–/– animals (Fig. 3, A and B). Overproduction of these inflammatory cytokines may contribute to liver disease. Of the panel of 16 factors evaluated, only the release of one proinflammatory cytokine, IL-1{alpha}, was significantly higher in TLR3–/– mice. Although its levels were similar when assayed on day 3, the elevated levels were sustained in the TLR3–/– mice before dropping to wild-type levels on day 5 (Fig. 3C, and data not shown). Among the other 13 cytokines examined, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, MCP-1, TNF-{alpha}, MIP-1{alpha}, GM-CSF, and RANTES, no significant differences were evident. However, as demonstrated in Fig. 3D, IL-12 levels were notably higher, albeit short of statistical significance, in the TLR3–/– mice on days 3 and 4 of infection.


Figure 3
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  		FIGURE 3. Differences in liver cytokine levels between PTV-infected wild-type and TLR3–/– mice. Day 0, 3, and 4 hepatic levels of IL-6 (A), IFN-{gamma} (B), IL-1{alpha} (C), and IL-12 (D), as measured by Q-Plex cytokine array. Each data point shown represents the value for a single mouse. *, p < 0.05; **, p < 0.01 compared with wild-type mice by Student’s t test.

 
Systemic IL-6 production is greatly reduced in TLR3–/– mice during PTV infection

Serum cytokine profiles were also obtained to assess whether a pronounced systemic response in wild-type animals may be contributing to increased susceptibility to PTV infection. As seen in the liver, wild-type animals produced remarkably more systemic IL-6 compared with TLR3–/– mice (Fig. 4A). Elevated mean levels of IL-6 were detected on both days 3 and 4 of infection that were 4- and 21-fold greater, respectively, than amounts produced by PTV-infected TLR3–/– mice. Day 4 levels of MCP-1 and RANTES were also found to be greatly reduced in TLR3–/– mice as compared with the wild-type animals (Fig. 4, B and C). The differences resulted from an overall decrease in cytokine levels from day 3 to 4 in the TLR3–/– mice as opposed to the high levels sustained by the wild-type mice. In contrast, and consistent with the liver data, IL-12 was found to be elevated in the TLR3–/– mice as compared with wild type. All four animals had significantly higher values on day 4, and with the exception of one mouse that presented with IL-12 below baseline levels, a similar trend was seen on day 3 (Fig. 4E). Of the other cytokines and chemokines analyzed, the only notable difference in serum levels between TLR3-deficient and wild-type mice was found with IL-10. Although not statistically significant, this cytokine was somewhat elevated in wild-type mice (Fig. 4D). It is important to note that the day 4 serum cytokine profiles obtained for the wild-type mice lacked the contribution of one to two of the four animals in the day 4 sacrifice group as a consequence of death (one mouse) and severe illness (one mouse), which limited serum collection. Thus, it is possible that the observed differences could have been greater because it is likely that extremely ill mice would present with highly elevated levels of many proinflammatory cytokines.


Figure 4
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  		FIGURE 4. Reduced circulating levels of IL-6 and other inflammatory mediators in PTV-infected TLR3–/– mice. Day 0, 3, and 4 systemic levels of IL-6 (A), MCP-1 (B), RANTES (C), IL-10 (D), and IL-12 (E) as measured by Q-Plex cytokine array. Due to mortality and severe illness of several mice at the time of sacrifice, the day 4 serum data are derived from three mice, with the exception of IL-10, for which data were only obtained for two mice due to shortage of serum from the extremely ill mouse. Each data point shown represents the value for a single mouse. *, p < 0.05; **, p < 0.01 compared with wild-type mice by Student’s t test.

 
IL-6–/– mice are highly susceptible to PTV infection

IL-6 is one of a number of inflammatory mediators that are believed to contribute to refractory shock and subsequent death associated with infection by various hemorrhagic fever viruses (24). Levels of this cytokine were profoundly elevated in both liver and serum samples from wild-type mice compared with those from TLR3–/– mice. Because IL-6, by far, resulted in the most dramatic difference seen by cytokine and chemokine profiling, we next evaluated its contribution to the increased mortality and liver disease observed with wild-type mice. We hypothesized that overproduction of IL-6 is detrimental to the outcome of PTV infection and examined whether mortality and disease parameters could be reduced in IL-6-deficient mice. As shown in Fig. 5, IL-6–/– mice were significantly more susceptible to PTV challenge than wild-type animals. In contrast to a mortality rate approaching 70% for the wild-type mice, there was 100% lethality in the IL-6–/– group. Consistent with the survival data, day 3 systemic and hepatic virus titers and serum ALT levels were markedly higher in the IL-6–/– mice (Fig. 6). Although liver scores (hepatic icterus) were greater in IL-6–/– mice (3.0 ± 0.8) as compared with wild-type mice (2.6 ± 1.1), the difference was not statistically significant. The data indicate that IL-6 is an indispensable component of the protective immune response to PTV.


Figure 5
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  		FIGURE 5. IL-6–/– mice demonstrate increased susceptibility to PTV infection. IL-6–/– (n = 10) and wild-type mice (n = 18) were challenged with PTV and observed for mortality on a daily basis for a duration of 14 days. Survival curves shown are representative of two similar experiments. *, p = 0.014 compared with wild-type mice.

 

Figure 6
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  		FIGURE 6. IL-6–/– mice have increased viral burden and liver damage on day 3 of PTV infection. Serum and liver virus titers (bars, left y-axis) and ALT release (diamond symbol, right y-axis) were measured 3 days post-PTV challenge. The limits of detection for the virus titer assays were as described in Fig. 3. ALT activity present in serum serves as an indirect measure of liver injury. Values represent the means and SDs of groups of five mice and are representative of two similar experiments. *, p < 0.05 compared with wild-type mice by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The resistance of TLR3–/– mice to PTV challenge was unanticipated considering most of the previously reported in vivo studies with TLR3-deficient mice. Of these investigations attempting to establish a protective role for TLR3 in host defense against lymphocytic choriomeningitis virus, vesicular stomatitis virus, mouse CMV (MCMV), reovirus, respiratory syncytial virus, and West Nile virus (WNV), significant differences in survival outcome were only observed with the latter mouse infection model (4, 6, 7, 8). Notably, however, Tabeta et al. (7) did report a very slight survival impairment with MCMV-infected TLR3–/– mice and also demonstrated markedly increased splenic viral loads in TLR3-deficient animals. In the WNV model, increased survival of TLR3–/– mice was demonstrated and found to be attributable to the lack of TLR3-dependent inflammation that facilitates viral entry into the brain, leading to greater lethality in wild-type mice due to neurological complications (8). Although the WNV results are consistent with our findings in the context of survival, caution must be taken when comparing the results due to the differences between the two models in disease manifestation and cause of death. Notably, systemic levels of WNV were found to be comparably higher in TLR3–/– mice than in wild-type animals (8). In contrast, PTV infection in mice, characterized as an acute fatal hepatic disease, resulted in a comparable spike in serum virus titers 2 days postinfection for both TLR3–/– and wild-type mice. Although high titers persisted several more days in the wild-type mice, viral burden in TLR3-deficient animals began to drop on day 3 and was comparably less by day 4. Despite clear indication of amplified liver disease in the wild-type animals as measured by serum ALT, infectious liver viral load was not appreciably different when compared with TLR3–/– mice. Thus, taking into consideration previous findings in which levels of proinflammatory cytokines were significantly diminished in TLR3–/– mice following challenge with MCMV and WNV (7, 8), our initial findings suggested that an exaggerated TLR3-mediated inflammatory response may be augmenting PTV pathogenesis and negatively affecting survival outcome and magnitude of hepatic disease. A thorough investigation into acute pneumonia caused by influenza A virus (IAV), published after the initial submission of our work, is in accord with our findings, supporting the notion of exuberant TLR3-mediated cytokine and chemokine responses negatively impacting survival outcome and disease severity during viral infection (25). As was the case in the PTV infection model, the advantage conferred by the absence of TLR3 in limiting IAV-induced disease and fatality was unanticipated.

Although cytokine and chemokine responses are critical to the coordination of the host immune response, excessive levels, as in the case of sepsis, can have profound toxic effects often leading to death. It is believed that disproportionate antiviral proinflammatory responses following infection with viral hemorrhagic fever agents contribute to terminal shock and defective coagulation associated with fatal infections (26). RVFV is known to cause severe disease in the form of hemorrhagic fever in a small percentage of human cases. As a model of severe RVFV infection and disease, PTV infection induced several inflammatory mediators that were, for the most part, elevated in infected wild-type mice compared with TLR3–/– mice. IL-6, IFN-{gamma}, MCP-1, RANTES, and, to a lesser degree, IL-10 were present at higher levels in wild-type PTV-infected mice. Concurrent with our findings, IL-6 expression was also found to be appreciably reduced in TLR3–/– mice after infectious challenge in the WNV model of lethal encephalitis (8). Moreover, IL-6, MCP-1, and RANTES were all notably reduced in bronchoalveolar lavage fluids from IAV-infected TLR3–/– mice (25). Interestingly, these mice presented with elevated levels of IFN-{gamma}, whereas TLR3–/– mice infected with PTV had reduced amounts of this cytokine in liver tissue, but not systemically when compared with wild-type mice. The differences in the levels of the above inflammatory mediators in the PTV infection model were not likely due to viral load because liver burden was comparable between the two strains of mice and serum levels reached equivalent peak titers.

It has been suggested that after the initial thrust in cytokine release in response to Ebola virus infection, a decrease to normal levels results in individuals who fail to develop clinical symptoms (27). This reduction is believed to minimize the toxic and often lethal effects that can arise from excessive proinflammatory cytokine production during infection with hemorrhagic fever-causing viruses. Consistent with this notion, significant differences in the levels of IL-6, IFN-{gamma}, MCP-1, and RANTES on day 4 of infection were due to increased or sustained release by the wild-type animals compared with the TLR3–/– mice, in which the levels dissipated after day 3. The exceptions to this were liver IL-1{alpha} and serum IL-12 levels, which were greater in TLR3–/– mice. Our data suggest IL-1{alpha}, a functionally pleiotropic cytokine that helps mediate the acute phase reaction in liver tissue in response to infection or trauma, may be negatively regulated by TLR3. Increased day 4 levels of IL-1{alpha} present in TLR3–/– mice may be highly beneficial in the PTV infection model, or at minimum, do not appear to negatively influence disease outcome. The same may be said for IL-12, a Th1-promoting proinflammatory cytokine critical to host antiviral defense (28). Notably, we did not detect remarkable levels of TNF-{alpha}, a major factor associated with septic shock induced by bacterial toxins and, in the case of WNV infection, one of the key cytokines that induces death (8). Our findings provide insight into the balance of cytokine and chemokine responses that promote survival after PTV infection while minimizing liver disease. Seemingly, the apparent dysregulation of the inflammatory response in mice lacking TLR3 serves to better limit immune-mediated hepatic insult, resulting in significantly increased survival.

It is widely accepted that IL-6 acts in a proinflammatory capacity, yet recent evidence would suggest that it also possesses immunosuppressive activity (29). Many of the inflammatory mediators, including IL-6, released in response to PTV infection are reportedly induced during human and nonhuman primate viral hemorrhagic fever infection (24). We postulated that IL-6 overproduction was contributing to the increased mortality observed in wild-type mice. Indeed, the association of exaggerated IL-6 production and lethal septic shock has been established (30, 31), and IL-6 and RANTES are reported to be elevated in cases of dengue hemorrhagic fever (32). Furthermore, herpes virus infections, although relatively mild in adults, can result in severe disseminated infections in neonates that clinically resemble bacterial sepsis. Evidence of exuberant IL-6 responses in neonates to HSV-1 suggests that elevated levels of this and other cytokines may contribute to severe forms of disease (33). To investigate the contribution of IL-6 to lethality caused by PTV infection, IL-6–/– mice were used and found to be significantly more susceptible to infection than their wild-type counterparts. Unfortunately, due to critical host defense role of IL-6 in the PTV infection model, a fair assessment could not be made in IL-6–/– mice because TLR3–/– animals did produce measurable levels of IL-6 following infection. Therefore, interpretation of these data in the context of IL-6 as a factor in viral pathogenesis should be approached with caution.

We propose that IL-6, which is involved in various aspects of antiviral defense including B cell maturation, CTL function, and the induction of acute-phase reaction mediators in hepatocytes (34), is needed initially to assist in the coordination of immune response. Considering that complete ablation of IL-6, opposed to controlling its production as seen in TLR3–/– mice, proved to be deleterious to survival and disease outcome, studies are being designed to attempt to neutralize the effects of IL-6 at later stages of infection with mAb. This approach will require considerable effort to identify the most appropriate amounts and time(s) of treatment that will produce IL-6 profiles similar to those seen in the TLR3-deficient mice. In addition, therapeutic intervention with drugs that target the NF-{kappa}B pathway and limit proinflammatory cytokine production, including IL-6, will be assessed to determine whether disease severity can be limited by such an approach. Also, studies monitoring inflammatory mediators during the course of fatal and nonfatal infections in wild-type mice will be done to correlate levels of IL-6 and other factors with disease outcome. The results of such studies may provide a blueprint for the type of cytokine and chemokine profiles that are conducive to survival in normal mice.

To our knowledge, this is the first report examining the role of a TLR family member in host defense against phleboviral infection. Our findings are not without precedent in the context of the TLR family of pattern recognition receptors. In addition to TLR3 studies with WNV and IAV discussed above (8, 25), similar results have been reported with other TLRs and viral and bacterial pathogens (35, 36). Notably, infection of TLR2–/– mice with HSV-1 did not result in dramatic differences in viral burden compared with wild-type mice; however, decreased levels of IL-6 and MCP-1 were associated with limited disease and reduced mortality (36). The idea that mice lacking a receptor that recognizes a prominent viral molecular pattern are more resistant to infectious challenge is somewhat perplexing. It is conceivable that in the event of naturally acquired viral infections, in which exposure to smaller infectious doses would occur, TLR3 may play a more significant role in managing infection and more effectively coordinating the balance of inflammatory mediators. By comparison, larger infectious doses may lead to overzealous proinflammatory responses in certain infection models that may be toxic and lead to death. Current views suggest that disproportionately high levels of proinflammatory cytokines are interconnected with lethal disease associated with viral hemorrhagic fever infections (26). Our findings with TLR3-deficient mice suggest that overproduction of IL-6 in liver tissue, as well as systemically, may contribute to the pathogenesis of PTV, and that controlled production, as is seen with TLR3–/– mice, is associated with a more favorable survival outcome.


   Acknowledgments
 
We thank Scott Kobayashi, John Morrey, and Heather Greenstone for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Contract NO1-AI-15435 from the Virology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Brian B. Gowen, Institute for Antiviral Research, Utah State University, 5600 Old Main, Logan, UT 84322. E-mail address: bgowen{at}cc.usu.edu Back

3 Abbreviations used in this paper: RVFV, Rift Valley fever virus; ALT, alanine aminotransferase; IAV, influenza A virus; MCMV, mouse CMV; PTV, Punta Toro virus; WNV, West Nile virus. Back

Received for publication May 17, 2006. Accepted for publication August 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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