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We investigated whether Toll-like receptor 3 (TLR3) stimulation would protect the host from inhaled Francisella tularensis. TLR3 is expressed by respiratory epithelial cells and macrophages and can be activated by a synthetic double-stranded RNA ligand called polyinosine-polycytosine [poly(I:C)]. Thus, we evaluated poly(I:C) as a novel treatment against inhaled F. tularensis. In vivo, BALB/c mice intranasally (i.n.) treated with poly(I:C) (100 μg/mouse) 1 h before or after Schu 4 or LVS (100 CFU) i.n. challenge showed that poly(I:C) treatment significantly reduced bacterial load in the lungs (P < 0.05). Bronchoalveolar lavage from poly(I:C)-treated mice alone or combined with F. tularensis infection significantly increased cytokine secretion and enhanced neutrophil influx to lung tissues. Poly(I:C) responses were transient but significantly prolonged the survival of treated mice after i.n. F. tularensis challenge relative to mock treated animals. This prolonged survival providing a longer window for initiation of levofloxacin (LEVO) treatment (40 mg/kg). Animals treated with poly(I:C), challenged with F. tularensis, and then treated with LEVO 5 days later had 100% survival relative to 0% survival in animals receiving LEVO alone. Mechanistically, poly(I:C) given to human monocyte-derived macrophages before or after Schu 4 or LVS challenge (multiplicity of infection, 20:1) had significantly reduced intracellular bacterial replication (P < 0.05). These data suggest that poly(I:C) may represent a potential therapeutic agent against inhaled F. tularensis that prolongs survival and the opportunity to initiate standard antibiotic therapy (i.e., LEVO).
Inhalation is likely to be one of the primary routes by which a bioweapon will be delivered to a target population. Francisella tularensis is a potential bioweapon because it can be aerosolized due to its inherently hardy nature, and less than 20 inhaled organisms can be detrimental to the host (8, 10, 28). F. tularensis, the etiologic agent of tularemia, is a small, Gram-negative nonmotile coccus and a facultative intracellular bacterium (16, 36). There are two major subspecies of F. tularensis; one is designated type A and includes strains that induce aggressive pathologies in the host and can result in pulmonary tularemia causing death if not treated (18, 37). The commonly studied virulent type A strain, Schu 4, was isolated originally from a human case of tularemia (22, 31, 38). The type B subspecies of F. tularensis causes a milder disease in humans than do type A strains. The only vaccine against tularemia known at this time was derived from subspecies type B and is called the live vaccine strain (LVS) (3, 8, 19, 31). Interestingly, most animal modeling of F. tularensis infection has used LVS-infected mice because it mimics the human disease caused by type A strains (6, 7, 9, 12). The immunological efficacy of LVS in humans is not known; vaccination with LVS does not provide complete protection against the virulent type A strains of F. tularensis (7, 19). As a result, alternative intervention strategies and vaccines need to be developed.
Ideally, vaccines against bioweapons will be established to protect the general population limiting the impact of such terroristic acts. Until such vaccines are available and widely distributed, alternate methods of broad range protection must be investigated. One interesting strategy is to engender innate immune resistance against mucosal pathogens (13, 17, 24). We have investigated the potential of Toll-like receptor (TLR) agonists recognized by TLRs highly expressed by respiratory epithelial cells. Specifically, polyinosine-polycytosine [poly(I:C)] is a synthetic double-stranded RNA analog that stimulates TLR3 triggering the induction of the host innate immune response including as RANTES, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 17, 23, 26).
Poly(I:C) can be delivered easily by a nose spray, is cheap to manufacture, and could be offered as an over-the-counter product unlike antibiotics. Importantly, the kinetics of cytokine secretion after poly(I:C) administration showed a transient response and offered no indication of toxicity, even with repeated use in our previous study (17). We therefore examined poly(I:C) as a topical treatment for a potential F. tularensis aerosol release. Theoretically, intranasal (i.n.) poly(I:C) could engender an innate immune response against F. tularensis, providing an extended period of resistance before an antibiotic, such as levofloxacin (LEVO), can be administered. LEVO belongs to the group of antibiotics known as fluoroquinolones and has been used to treat respiratory infections such as tularemia (1, 21, 25).
Recently, we established that genital application of poly(I:C) protected against lethal HSV-2 challenge in mice (17). We have extended these findings by applying poly(I:C) to the respiratory mucosa testing the hypothesis that nucleic acid-based TLR agonists may prove to be useful prophylactic and possibly therapeutic measures against select agent respiratory infections including F. tularensis. Because F. tularensis suppresses the innate immune response (2, 4, 29, 39), the host does not detect and/or respond to the organism for approximately 48 to 72 h after F. tularensis infection (2; T. D. Eaves-Pyles, unpublished data). This large gap between the time of infection and host detection of the organism limits the development of an adequate immune response against F. tularensis. As such, we hypothesized that poly(I:C) would enhance the host's response prior to or soon after F. tularensis exposure. Our in vivo and in vitro studies show that mice treated 1 h before or 1 h after the administration of poly(I:C) had significantly less bacteria in their lungs, increased neutrophil infiltration to the lung, and extended survival after LVS or Schu 4 infection. Moreover, mice treated with poly(I:C) (1 h after Schu 4 infection), followed by LEVO administration 5 days later, were fully protected from lethal outcomes. Corresponding to these in vivo studies, we show that poly(I:C)-treated human monocyte-derived macrophages (MDM) secreted high levels of specific cytokines and engendered enhanced intracellular bacterial killing after LVS and Schu 4 exposure compared to untreated animals.
Schu 4 (U.S. Army DPG, Life Sciences Division, Dugway, UT) and LVS (ATCC 29684) were cultured as previously described (14). Briefly, bacteria were grown overnight at 37°C in Mueller-Hinton broth II (Difco Laboratories, Detroit, MI) supplemented with IsoVitaleX (Becton Dickinson, Cockeysville, MD) from frozen stocks. The number of bacteria was determined by using a Petroff-Hauser counting chamber (Hausser Scientific, Horsham, PA). Bacterial serial dilutions were plated on cysteine heart agar (CHA) plates supplemented with IsoVitaleX to confirm experimental dosage.
Human monocytes were isolated from buffy coats from healthy donors provided by the University of Texas Medical Branch (UTMB) Blood Bank. Isolation utilized a combination of Ficoll density gradient centrifugation (800 × g at 21°C) and counterflow centrifugal elutriation (Beckman J2-21 M/E centrifuge with JE-B6 elutriator rotor; Beckman Instruments, Palo Alto, CA). The monocytes were cultured in RPMI 1640-10% fetal bovine serum supplemented with granulocyte-macrophage colony-stimulating factor for 7 days to ensure full macrophage differentiation. Cultures were evaluated by flow cytometry to confirm differentiation and were found to be >95% macrophage phenotype (data not shown). After poly(I:C) treatment and/or LVS or Schu 4 (multiplicity of infection [MOI], 20:1) challenge, MDM supernatants were collected 2 h postinfection and analyzed for cytokine secretion via enzyme-linked immunosorbent assay (ELISA). MDM then were treated with gentamicin (50 μg/ml) for 30 min to kill extracellular bacteria and washed twice with phosphate-buffered saline (PBS). A portion of each MDM culture was lysed with 0.1% sodium dodecyl sulfate (SDS) and then plated on CHA plates for 48 h. F. tularensis colonies were counted to determine MDM phagocytosis. Remaining MDM were resuspended in fresh RPMI and incubated overnight. The next day (18 h), MDM were treated with gentamicin, lysed, and plated as described above. CFU were determined from these cultures to represent intracellular replication or killing of F. tularensis.
Six-week-old female BALB/c mice weighing between 20 and 22 g (Jackson Laboratories, Bar Harbor, ME) were housed in an Association for Assessment and Accreditation for Laboratory Animal Care-approved housing facility and allowed to acclimate for 7 days prior to procedures receiving free access to food and water. All procedures were approved by the UTMB IACUC and performed humanely with minimal suffering. The animals were anesthetized with pentobarbital and restrained vertically using sterilized commercial fishing line looped behind the upper incisors and connected to a support platform. A total of 20 μl containing 100 μg of poly(I:C), poly(I) (Sigma Chemical Company, St. Louis, MO) or PBS, and/or PBS containing LVS or Schu 4 were placed at the anterior of each nares (10 μl/nare), and the animals inhaled the solutions naturally. Stocks of poly(I:C) or poly(I) were established in sterile PBS (CellGro Mediatech, Manassas, VA) and were confirmed to be free of contaminating lipopolysaccharide by testing in HEK-293 cells expressing TLR3 (positive), TLR4, or TLR5 (negative), as well as mixing with polymyxin B (data not shown). The bacterial challenge varied depending on the experiment and is noted in the results and figure legends. After i.n. administration, the animals were placed back in their cage and allowed to recover. LEVO was administered by intraperitoneal (i.p.) injection of 40 mg/kg 5 days after poly(I:C) treatment. PBS was used as a control.
For bacterial replication studies the animals were sacrificed humanely by pentobarbital overdose. The lungs were harvested 24 h posttreatment and infection and homogenized with glass stoppers in 2 ml of sterile PBS in a sterile culture hood. The supernatants then were plated in duplicate on CHA plates for enumeration of viable LVS organisms. The replication of the organism was compared to animals treated with the PBS vehicle alone. The bacterial challenge inoculum also was plated to ensure viability and organism numbers.
The collection of mouse BAL was performed by sacrificing the mice with an overdose of pentobarbital, the trachea was then exposed, and a small incision was made to insert a 24-gauge round-ended gavage needle attached to a 1-ml tuberculin syringe containing 700 μl of PBS supplemented with 5 mM EDTA (pH 7.2). BAL was performed with 700-μl aliquots until a total lavage volume of 1.5 ml was collected. The BAL fluid was collected by slowly withdrawing the fluid from the lungs and placing it in cold polystyrene tubes containing protease inhibitors. All tubes were kept on ice during BAL collection. The cells were separated by centrifugation (300 × g for 10 min), and the supernatants were analyzed for cytokines by ELISA (Pierce/Endogen).
Cells collected from each BAL were resuspended in PBS containing 10% fetal calf serum and 0.02% sodium azide. The cells then were incubated with Fc block for 5 min., followed by a 30-min incubation with the following antibodies to label neutrophils: anti-CD11c (phycoerythrin [PE] or allophycocyanin), anti-CD11b (PE-Cy5 or allophycocyanin-Cy7), and anti-GR-1 (Pey7). Antibodies were purchased from Serotec, BD Pharmingen, or eBioscience, unless otherwise indicated. Cells were washed in PBS and then resuspended in 2% paraformaldehyde and analyzed by the UTMB Flow Cytometry Core on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). The cells were gated according to their forward- and side-scatter properties, as well as pan-markers.
Experimental conditions were performed in triplicate (in vitro) or utilized groups of 5 or 10 mice and were repeated to confirm the findings and to increase statistical confidence. Survival data were evaluated by log-rank analyses with Welch's correction using Prism software (v 4.0; GraphPad, San Diego, CA). The in vitro studies were repeated with cells from other donors with indistinguishable outcomes. Also using Prism software, differences among in vitro experimental conditions and controls were compared by analysis of variance, followed by Tukey's test or Bonferroni's multiple-comparison test. A P value of <0.05 was considered significant.
The longer the delay in the start of standard intervention against F. tularensis, the less effective “front line” antibiotics are against an infection (21). Further, agents to protect at risk populations from F. tularensis should be employable when forewarning is available or just after exposure. To evaluate the potential of poly(I:C) as such an agent, we tested delivery prior to or just after F. tularensis inoculation in mice. Outcomes addressed included pathogenesis and bacterial replication levels in the lung.
To determine whether poly(I:C) could alter the replication rate and/or delay the onset of inhalation tularemia in the mouse model, we first delivered 100 μg i.n. at selected times prior to or just after challenge. Time points were selected to define the window of opportunity before or after i.n. administration and tested as single poly(I:C) doses in BALB/c mice. The results showed that poly(I:C) delivered at 24 h (P < 0.01), 1 h (P < 0.001) before or 1 h after (P < 0.01) LVS challenge significantly reduced the bacterial load in the lungs 24 h postinfection compared to PBS controls (Fig. (Fig.1).1). Interestingly, mice treated 1 h prior to challenge with 100 CFU had the lowest detectable LVS in their lungs compared to all other groups (P < 0.01). Mice treated 1 h after bacterial challenge had significantly reduced bacterial titers relative to PBS-treated mice (Fig. (Fig.1;1; P < 0.01).
In a follow-up study, additional poly(I:C) application times were evaluated relative to application of the PBS vehicle alone. This study confirmed the results and also evaluated protection against a higher bacterial challenge (1,000 CFU; Fig. Fig.1,1, right bars). Again, relative to PBS-treated control mice, a significant reduction in bacterial replication was afforded by poly(I:C) application (P < 0.05) 24 h prior to challenge. These results suggested that poly(I:C) reduced the F. tularensis bacterial load in the lungs before and after LVS infection.
To determine the effects of poly(I:C) on the lungs and provide a likely explanation for the observed bacterial load reduction, cytokine secretion was quantified in mouse BAL. BALB/c mice were treated with 100 μg of poly(I:C), 100 μg of poly(I), PBS, or 100 CFU of LVS, followed by BAL at 24, 48, or 72 h posttreatment or postinfection. The results showed that the selected cytokine levels in the BAL of poly(I:C)-treated mice were considerably higher than PBS-treated, poly(I)-treated, or LVS-infected mice at 24 h posttreatment with significantly higher amounts of IL-6, MCP-1, and IL-1α (Fig. (Fig.2A;2A; P < 0.05). The cytokine levels remained elevated in BAL 48 h after poly(I:C) treatment (Fig. (Fig.2A;2A; IL-1α, MCP-1, and tumor necrosis factor alpha [TNF-α]; P < 0.05) compared to the PBS- and poly(I)-treated controls. Cytokines in the BAL of LVS-infected mice did not increase until 48 h postinfection in a pattern distinct from poly(I:C) (Fig. (Fig.2A).2A). IL-6 levels in LVS-infected mice were significantly higher than poly(I:C)-treated mice at 48 h, with IL-1α, IL-6, and TNF-α being significantly elevated in LVS-infected mice compared to the PBS and poly(I) controls (Fig. (Fig.2A;2A; P < 0.05). By 72 h the levels of cytokines in LVS-infected mice were considerably higher compared to other groups, with significantly higher levels of MCP-1 and TNF-α relative to all other groups (P < 0.05). Only minimal cytokine levels were detected 72 h after poly(I:C) treatment (Fig. (Fig.2A),2A), indicating the transient nature of the response. The PBS and poly(I) negative controls did not induce cytokine above basal levels at any of the tested time point (Fig. (Fig.2A2A).
In addition, the influx of neutrophils in the BAL was significantly higher at 24 and 48 h in poly(I:C)-treated mice compared to all other groups (Fig. (Fig.2B;2B; P < 0.05), followed by a subsequent drop to nearly undetectable levels by 72 h. Conversely, neutrophils were not detected in the lungs of LVS-infected mice until 72 h postinfection where neutrophils in the BAL were significantly higher than all other groups (Fig. (Fig.2B;2B; P < 0.05).
Quantification of cytokines in the BAL of mice treated with poly(I:C) before (1 h) or after (1 h) LVS challenge (10 50% lethal doses or 100 CFU/mouse) showed that the cytokine secretion levels were highest at 24 h both 1 h before and 1 h after in poly(I:C)-treated mice infected with LVS (Fig. (Fig.3;3; IL-1α, IL-6, MIP-1α, and IL-8) compared to 48 and 72 h. This was followed by a steady decline in cytokine secretion over time that reached basal levels by 72 h. Poly(I:C) treatment 1 h before or 1 h after infection was equally effective at inducing cytokine secretion. Conversely, i.n. application of PBS 1 h before (Fig. (Fig.3A)3A) or after LVS (data not shown) had little to no effect on the host's ability to induce cytokine secretion in the lungs at any of the tested time points (Fig. (Fig.3A)3A) compared to poly(I:C)-treated mice before and after LVS infection (Fig. (Fig.3A).3A). Because no differences were observed in the levels of cytokine secretion in mice receiving PBS before or after infection, only the results for PBS given 1 h preinfection are shown in Fig. Fig.3A.3A. More important is that at 24 h after LVS infection there was significant neutrophil migration to the lungs of mice receiving a pre- or posttreatment of poly(I:C) compared to PBS-treated/LVS-infected mice (Fig. (Fig.3B3B).
These culminated findings demonstrate that the host does not respond to F. tularensis for 48 to 72 h postinfection. However, mice treated with poly(I:C) 1 h pre- or postinfection resulted in earlier cytokine secretion in the lungs and neutrophil migration to the lungs by 24 h, which likely provided the host with an opportunity to mount a more effective innate immune response against F. tularensis.
As determined above, poly(I:C)-treated mice elicited an innate immune response that appeared sufficient to reduce the bacterial load of F. tularensis in the lung. To expand on these findings, mice were pretreated i.n. with 100 μg of poly(I:C) 24 or 1 h preinfection or 1 h postinfection with 100 CFU of LVS per mouse (n = 10 mice per group). Control mice were given the PBS vehicle. Survival was observed for 20 days posttreatment and infection. The results showed that survival was significantly extended when mice were treated with poly(I:C) compared to LVS-infected mice alone (P < 0.05; Fig. Fig.4A).4A). Every infected, untreated mouse died by day 6 postinfection. However, the 24 h pretreatment or 1 h posttreatment of poly(I:C) extended mouse survival by an average of 3 days (Fig. (Fig.4A).4A). Poly(I:C) delivered 1 h prior to infection was the most effective treatment tested, increasing survival by an average of 8 days (Fig. (Fig.4A,4A, P < 0.0004).
Survival studies also were completed by testing additional time points of poly(I:C) treatment in mice infected with the virulent Schu 4 F. tularensis human isolate. The results showed that Schu 4 led to lethal disease in all PBS-treated animals by day 7 postinfection (Fig. (Fig.4B).4B). Similar to the findings with LVS-infected mice, 1 h post-poly(I:C) treatment delayed lethal disease by an average of 2 days relative to PBS treatment (Fig. (Fig.4B).4B). Remarkably, poly(I:C) administered 1 h preinfection not only extended mouse survival but also protected some animals up to 15 days postinfection (Fig. (Fig.4B;4B; P < 0.05). Interestingly, when poly(I:C) and Schu 4 were administered simultaneously, mouse survival was extended 8 days longer than Schu 4-infected mice receiving PBS (Fig. (Fig.4B;4B; P < 0.05).
To address the potential for poly(I:C) to extend the period before successful antibiotic treatment is initiated, we coupled 1-h post-poly(I:C) treatment with LEVO, which is considered to be a “gold standard” antibiotic treatment for F. tularensis.
Treatment with poly(I:C) 1 h after F. tularensis challenge was selected because it showed some protection but was not as effective as the other treatments. Importantly, this time point also reflects a postexposure, therapeutic regimen germane to a real-world scenario. Our results showed that LEVO initiation at 5 days postchallenge was ineffective against F. tularensis challenge (Fig. (Fig.4B).4B). To determine whether poly(I:C) could extend the time before effective LEVO therapy initiation, mice were treated with poly(I:C) 1 h post-Schu 4 challenge and then, 5 days later, they were i.p. injected with 40 mg of LEVO/kg (21). This study also repeated groups of animals treated with poly(I:C), followed by Schu 4 infection, to confirm the previous results. As noted, substantial increases in survival times were again observed. In the group treated with poly(I:C) 1 h after Schu 4 challenge and LEVO 5 days later, every animal recovered and survived until euthanization on day 15 (Fig. (Fig.4B).4B). This is in dramatic contrast to our previous findings that showed LEVO therapy initiated at 5 days postchallenge did not resolve lethal F. tularensis infection (21). Importantly, these increased survival times also allowed for successful initiation of LEVO treatment even 5 days after Schu 4 challenge (Fig. (Fig.4B).4B). Tissues from these animals showed no viable Schu 4 (data not shown). Together, the data support the concept that even postexposure, therapeutic poly(I:C) could provide a longer window of opportunity to initiate antibiotic treatment.
In addition to neutrophils, macrophages are key mediators of innate immunity and likely contribute to the responses of the i.n.-delivered poly(I:C). Because F. tularensis replicates in alveolar macrophages and suppresses macrophage cytokine secretion to ensure its survival (2, 5, 29), we examined poly(I:C) treatment of MDM, following F. tularensis infection, for their ability to secrete cytokines, phagocytosis, and/or F. tularensis killing. Human MDM (106 MDM) were treated with poly(I:C) (100 μg/ml) at 24 or 1 h preinfection or 30 min postinfection with LVS or Schu 4 (MOI = 20:1). Parallel controls were mock treated with PBS. After 2 h of incubation, MDM were treated for 30 min with 50 μg of gentamicin/ml to kill extracellular bacteria, and then MDM lysates were plated to determine intracellular F. tularensis. These samples were designated time zero cultures and illustrated phagocytosis. Parallel MDM cultures were treated as described above but, after gentamicin treatment, the antibiotic was removed, and the MDM cultures were incubated overnight. The next day, intracellular F. tularensis was determined as described above.
Both types of F. tularensis tested address possible bacterial strain effects. The results showed that Schu 4 was phagocytosed by MDM in higher numbers compared to LVS regardless of the poly(I:C) administration (Fig. (Fig.5).5). Replication of Schu 4 and LVS were significantly reduced in MDM pretreated with poly(I:C) 24 h or 1 h prior to infection (Fig. (Fig.5;5; P < 0.05). LVS replication continued to be significantly impaired in MDM treated with poly(I:C) 30 min postinfection (Fig. (Fig.5A;5A; P < 0.05). Interestingly, the 30 min after poly(I:C) treatment cultures were not effective against Schu 4, where high bacterial replication was observed in MDM similar to PBS-treated controls (Fig. (Fig.5B5B).
To determine whether poly(I:C) induced cytokine secretion from MDM, cell supernatants were collected from poly(I:C)-treated and from PBS-treated MDM 2 h after F. tularensis infection and then were analyzed for IL-8, TNF-α, and IFN-γ. The ability of F. tularensis to suppress the innate immune response was again indicated by the lack of cytokine secretion from untreated MDM infected with Schu 4 and to a lesser extent LVS (Fig. (Fig.6).6). Conversely, F. tularensis-infected MDM treated with poly(I:C) showed higher cytokine secretion compared to their counterparts receiving vehicle only (P < 0.05; Fig. Fig.6).6). These data suggest that poly(I:C) enhanced F. tularensis killing and induced the secretion of cytokines from MDM that allowed the host to respond to inhaled F. tularensis and fight against the pathogen in a timely fashion.
The results of these studies showed that i.n. administration of a synthetic TLR3 agonist, poly(I:C), induced an early and effective innate immune response that enhanced bacterial clearance in mouse lungs, augmented cytokine secretion, and improved survival in F. tularensis-infected mice. Further, poly(I:C) enhanced F. tularensis killing by MDM and increased cytokine secretion from MDM. Importantly, poly(I:C) effects were transient, suggesting a lack of chronic inflammatory outcomes after a single i.n. administration.
After inhalation, F. tularensis has developed strategies to manipulate host responses such that the pathogen suppresses pulmonary innate immunity subsequently allowing F. tularensis to go undetected by the host for days (Fig. (Fig.2).2). It has been shown that Schu 4-infected mice failed to secrete TNF-α and IL-12p40 until 48 h postchallenge, but the secretion of the immunosuppressive cytokine transforming growth factor β was detected in these mice (2). In addition, F. tularensis subverts the host immune response by avoiding phagolysosomal fusion, allowing escape from the phagosome and subsequent replication in the cytoplasm of the cell (5, 15, 29). F. tularensis also interferes with IFN-γ signaling in human and murine mononuclear phagocytes that increased intracellular F. tularensis survival (29). All of these strategies provide a permissive host environment in which the pathogen can survive, colonize, and replicate in the lung. Thus, by the time the host initiates an immune response, F. tularensis has colonized the lungs and has begun to disseminate to distal organs.
We hypothesized that a TLR3 agonist, poly(I:C), would prime respiratory mucosal innate immune responses increasing the resistance to infection by F. tularensis, serving as a novel method for protection against inhaled bioagents. Poly(I:C) afforded protection against a mouse model of genital herpes simplex virus type 2 (17). Other investigators have demonstrated that TLR4 (24) or TLR9 (12, 13) agonist application increased cytokine production and prolonged survival in F. tularensis inhalation (24) or i.p. challenge mouse models (12, 13). However, there are no reported studies that evaluated the effectiveness of poly(I:C) against pneumonic tularemia caused by LVS or the virulent human F. tularensis isolate Schu 4. The data reported here establish that poly(I:C) provided significant protection against F. tularensis respiratory challenge in BALB/c mice in a time-dependent fashion.
A single poly(I:C) application delivered i.n. significantly reduced F. tularensis replication in BALB/c mice after a bacterial challenge with 100 or 1,000 LVS organisms. This challenge is universally lethal without intervention. Mechanistically, reduction of bacteria in the lungs of poly(I:C)-treated/LVS-infected mice likely was potentiated by the early production of cytokines and chemokines, resulting in the timely recruitment of immune cells to the lungs. These results are comparable to those found in mice treated with the TLR4 agonist, AGP, 24 h before and 48 h after aerosolization or i.n. exposure to F. novicida (24).
Our data show that a single dose of poly(I:C) administered to mice 1 h preinfection or 1 h postinfection was sufficient to stimulate the release of a variety of cytokines, including IL-1α, IL-6, IL-8, MIP-1α, and TNF-α (Fig. (Fig.22 and and3).3). Each of these cytokines forms an important part of the host's inflammatory response against infection and are well-known activators, as well as mediators of the lung immune response. IL-1α activates lymphocytes by increasing the expression of adhesion factors on endothelial cells to enable transmigration of leukocytes (20). IL-6 can act as a proinflammatory cytokine that induces B cells differentiation and antibody production (20). A primary role of TNF-α is to stimulate immune cell antimicrobial activities (20). In addition, IL-8 and MIP-1α are powerful chemoattractants that recruit neutrophils and other immune cells to the site of infection. Correlating with the secretion of these cytokines was a significant increase in neutrophil migration to the lungs of LVS-infected mice within 24 h after poly(I:C) treatment. Conversely, neutrophil infiltration into the lungs of LVS-infected mice alone did not occur until 72 h postinfection.
Although neutrophils are one of the predominant and first immune cells recruited to a site of the infection, their role during an F. tularensis infection is not well understood and somewhat confusing as conflicting findings are reported in the literature (27, 30, 34, 35). For example, it has been shown that in vitro the less-virulent LVS is resistant to killing by human neutrophils (27). Other findings in vitro showed the oxidative defense mechanisms of neutrophils are disrupted by LVS (34). Thus, the sum of these data indicate that neutrophils do not play a vital role in the clearance of F. tularensis. Conversely, Sjostedt et al. (35) demonstrated in vivo that neutrophil depleted mice were killed rapidly by a sublethal intravenous or intradermal injection of LVS compared to normal mice. That same study showed that LVS replicated steadily over time in the lungs, livers, and spleens of neutrophil-depleted mice, reaching lethal numbers, but the infection was resolved in normal mice (35). Anecdotally, a lethal human tularemia infection was reported in a neutropenic bone marrow transplant recipient who suffered from prolonged neutropenia and was associated with F. tularensis sepsis (30). Collectively, these in vivo findings support an important role for neutrophils against F. tularensis in an intact biological system.
Although we did not directly observe the clearance of F. tularensis by neutrophils in our in vivo studies, there was a correlation between cytokine secretion, the migration of neutrophils to the lungs, and decreased bacterial load in the lungs of poly(I:C)-treated and F. tularensis-infected mice. Similarly, mice treated with a TLR4 agonist 24 h before and 48 h after exposure to F. novicida also had increased neutrophil migration to the lungs compared to untreated, F. novicida-infected mice (24).
Due to the uncertain role of neutrophils, the impact of poly(I:C) on MDM also was investigated. Our in vitro studies indicated that human MDM are better able to fight against an F. tularensis infection when treated with poly(I:C) based upon substantially reduced intracellular LVS or Schu 4 loads. Interestingly, poly(I:C) treatment reduced intracellular LVS replication at all poly(I:C) treatment times and Schu 4 replication at 24 and 1 h pretreatment with poly(I:C) but not at 30 min posttreatment with poly(I:C) compared to untreated F. tularensis-infected MDM. These data suggest that if F. tularensis can establish infection of MDM, there is a quick (<30 min) subversion of the innate immune response of this target cell that prevents poly(I:C) from being effective. Together with the in vivo data indicating multicellular lung tissues could be protected by poly(I:C) administration up to 1 h after bacterial challenge, it is clear that additional study of the evasion of immune responses by F. tularensis must be completed.
Similar to the mouse BAL outcomes, poly(I:C) treatment given at 24 or 1 h preinfection induced significant IL-8, IFN-γ, and TNF-α secretion from LVS- and Schu 4-infected MDM compared to untreated and F. tularensis-infected animals. These data support the mechanistic role for these cytokines in resistance to F. tularensis and indicated a IFN-γ-specific response to poly(I:C). IFN-γ plays a central role in modulating the host innate immune response through the upregulation of macrophage IFN-γRI and the production of reactive oxygen species and nitric oxide (32, 33). F. tularensis circumvents these immunoprotective strategies by interfering with IFN-γ signaling via the suppression of STAT1 expression and phosphorylation resulting in the suppression of IFN-γ-induced iNOS, allowing for intracellular survival and replication of the bacteria (29). The administration of exogenous IFN-γ intracellular reduced bacterial survival in a macrophagelike cell line when added 8 h prior to infection but not at 8 h postinfection (29). Most recently, Elkins et al. (13) reported that coculture of LVS-infected murine macrophages with splenocytes isolated from mice treated with the TLR9 agonist, CpG, controlled intracellular LVS growth. These results were reversed with the use of an antibody to IFN-γ or TNF-α (13). Together, our results and those of Elkins et al. (13) support the hypothesis that the induction of IFN-γ MDM enhances and/or contributes to intracellular F. tularensis killing.
Most importantly, we observed improved survival in poly(I:C)-treated and F. tularensis-infected mice compared to infected mice alone. Specifically, delivery of poly(I:C) 1 h after LVS or Schu 4 infection prolonged survival by 2 to 3 days. Optimal protection was afforded by administering poly(I:C) 1 h prior to challenge, which provided the best protection against 100-fold more LVS or Schu 4. Similar findings using an alternative route of TLR treatment and F. tularensis infection were reported in mice given an i.p. treatment of CpG 2 to 3 days prior to a lethal i.p. challenge with LVS that increased survival compared CpG administered intramuscularly and untreated controls (12). CpG treatment did not afford protection when given on the same day or 1 day later with an i.p. LVS challenge (12). Thus, the protective affects afforded by various TLR agonists is dependent of the time and route of administration before or after a bacterial challenge. However, treatment with individual TLR agonists i.n. does not provide sufficient protection against inhaled F. tularensis when used alone because the animals eventually succumb to the infection.
Based on our results, we believe poly(I:C) should be further evaluated as a frontline affordable, self-administered intervention just prior to or immediately after a release of aerosolized F. tularensis into the environment. The completed combination therapy study indicates poly(I:C) would provide additional time to seek more aggressive treatment, specifically with antibiotics, from a healthcare professional. The combination of poly(I:C) 1 h postinfection with a one-time dose of LEVO 5 days after Schu 4 challenge allowed every animal to resolve the infection and conferred 100% survival.
We thank Lynn Soong (UTMB), Lijun Xin (UTMB), and Jignesh Patel (UTMB) for their assistance with the neutrophil migration studies and analysis of the flow cytometry data.
This study was primary funded by NIH/NIAID grant R21 AI068774.
Editor: J. N. Weiser
Published ahead of print on 1 February 2010.