ABSTRACT
Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular, primarily pulmonary, bacterial pathogen. Although much is known about adaptive immune responses against this bacterium, our understanding of innate immune responses against C. burnetii is not well defined, particularly within the target tissue for infection, the lung. Previous studies examined the roles of the innate immune system receptors Toll-like receptor 2 (TLR2) and TLR4 in peripheral infection models and described minimal phenotypes in specific gene deletion animals compared to those of their wild-type controls (S. Meghari et al., Ann N Y Acad Sci 1063:161–166, 2005, http://dx.doi.org/10.1196/annals.1355.025; A. Honstettre et al., J Immunol 172:3695–3703, 2004, http://dx.doi.org/10.4049/jimmunol.172.6.3695) . Here, we assessed the roles for TLR2, TLR4, and MyD88 in pulmonary C. burnetii infection and compared responses to those that occurred in TLR2- and TLR4-deficient animals following peripheral infection. As observed previously, neither TLR2 nor TLR4 was needed for limiting bacterial growth after peripheral infection. In contrast, TLR2 and, to a lesser extent, TLR4 limited growth (or dissemination) of the bacterium in the lung and spleen after pulmonary infection. TLR2, TLR4, and MyD88 were not required for the general inflammatory response in the lungs after pulmonary infection. However, MyD88 signaling was important for infection-induced morbidity. Finally, TLR2 expression on hematopoietic cells was most important for limiting bacterial growth in the lung. These results expand on our knowledge of the roles for TLR2 and TLR4 in C. burnetii infection and suggest various roles for these receptors that are dictated by the site of infection.
INTRODUCTION
Coxiella burnetii, the causative agent for Q fever, is an obligate intracellular Gram-negative bacterial pathogen that most commonly is acquired through aerosol inhalation (1). Natural infection can occur from exposure to animals or animal products, such as unpasteurized milk. Although large outbreaks are rare, they still occur, with over 4,000 cases reported in a recent outbreak in the Netherlands, due primarily to close proximity to infected dairy goat operations (2). Acute infection presents with flu-like illness, including headache and neurological, gastrointestinal, endocrine, and renal symptoms (3, 4). The infection can progress to a chronic state in a small fraction of people, which has been associated with the development of endocarditis and hepatitis (5).
Two phase variants of C. burnetii (phase I and II) have been defined. The phase I form is the cause of Q fever, whereas phase II variants are selected from phase I isolates following serial passage in eggs or in vitro tissue culture. Although phase II variants are infectious, they are considered far less virulent. The major virulence determinant of C. burnetii is its lipopolysaccharide (LPS) (6). A larger LPS structure containing an O antigen is expressed on phase I isolates, whereas a truncated LPS is expressed on phase II C. burnetii. Due to the extreme infectivity of phase I variants (less than 10 viable organisms) (7, 8), stability in the environment, and aerosol route of transmission, C. burnetii is considered a potential biological weapon and is classified as a category B select agent by the Centers for Disease Control and Prevention. There is currently no Q fever vaccine approved by the FDA in the United States.
Alveolar macrophages and monocytes are believed to be the primary targets of infection for this pathogen (9, 10). Although much is known about the role of macrophages in C. burnetii infection, we have an incomplete understanding of the roles of Toll-like receptors (TLRs) in C. burnetii pathogenesis. TLRs are a family of pattern recognition receptors found on macrophages and other cells that are important in innate immune responses. TLRs sense distinct microbial structures/patterns, such as cell wall determinants or nucleic acid motifs, and are critically important in early innate immune cell responses against a variety of infections. MyD88 is an adapter protein that mediates signal transduction for most TLRs, leading to robust immune cell activation and inflammation (11). Macrophage recognition of bacterial pathogens involves multiple members of the TLR family, but two are particularly important in recognizing surface structures on bacteria. Specifically, TLR4 recognizes Gram-negative bacteria through the detection of LPS (12, 13), whereas TLR2 recognizes surface molecules on both Gram-positive and -negative bacteria (14–16). Both TLR4 and TLR2 signal an inflammatory response through MyD88 (17, 18), and MyD88-mediated signaling has been shown to be important for pulmonary host defense against several pathogens (19–21). TLR4 also triggers the TRIF pathway, leading to a type I interferon response (22). Studies on TLR4 and TLR2 in C. burnetii infection have been done in mice and in vitro using human cells (23–26). Phase I C. burnetii LPS is recognized by human TLR4, but the interaction does not drive a robust inflammatory response, although it does contribute to the uptake of the bacterium and filamentous actin reorganization (23, 26). Furthermore, although C. burnetii expresses TLR2 ligands, it is thought that phase I LPS masks the recognition of TLR2 ligands expressed by C. burnetii, which are exposed on phase II isolates (25).
One prevailing view concerning the role of TLRs in Q fever is that phase I C. burnetii invades cells without stimulating TLR2 and TLR4, which contributes to immune evasion (6). Past studies in TLR2- and TLR4-deficient animals support this view, since the clearance of phase I C. burnetii is not significantly altered in TLR2- and TLR4-deficient animals compared to that in wild-type mice, although both TLR2- and TLR4-deficient animals have reduced granuloma formation (24, 26). However, these in vivo studies are incomplete, since the disease course in the TLR-deficient animals was investigated following peritoneal infection, whereas C. burnetii is naturally a pulmonary pathogen. TLR expression and function on monocytes and macrophages differ between the lung and the periphery (27). We also have little insight into the role for MyD88 in C. burnetii infection. Thus, gaps in knowledge exist for C. burnetii infection, as TLRs and downstream signaling pathways have not been studied following a route of infection relevant to natural infection and pathogenesis. Such studies are critical to our understanding of C. burnetii pathogenesis.
In this study, phase I C. burnetii intratracheal (i.t.) challenge was examined in TLR4-, TLR2-, and MyD88-deficient mice and compared to peripheral (peritoneum) challenge in the same mouse lines. Contrary to the prevailing view on the role of TLRs in C. burnetii pathogenesis, differences in bacterial burden and disease were detected between deficient mice and wild-type control mice following pulmonary infection. These results expand on our understanding of TLR2 and TLR4 in C. burnetii infection in an important way and suggest distinct and various roles for these receptors that are dictated by the site of infection. Our studies illustrate the importance of conducting experiments using relevant routes and tissues for infection to better understand the role of key innate immune cell recognition receptors in pathogenesis.
MATERIALS AND METHODS
Ethics statement.All animal experiments were performed in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of Montana State University. All efforts were made to minimize animal suffering.
Bacterial strain, mice, and in vivo infection.A Coxiella burnetii Nine Mile phase I (NMI) strain (RSA493) was kindly donated by Robert Heinzen at Rocky Mountain Laboratories, Hamilton, MT. All use of C. burnetii was done in a CDC-certified biosafety level 3 (BSL-3) facility approved for category B select agent use. Mice used were 6- to 12-week-old females. TLR4−/−, TLR2−/−, and MyD88−/− mice were bred in the Animal Resource Center at Montana State University. Sex- and age-matched C57BL/6 wild-type mice were either from our animal facility or obtained from Charles River (North Franklin, CT) or The Jackson Laboratory (Bar Harbor, ME). TLR4−/−, TLR2−/−, MyD88−/−, and wild-type C57BL/6 mice were infected intratracheally (i.t.) (1 × 103, 1 × 104, or 1 × 105 genome equivalents [GE]) or intraperitoneally (i.p.) (1 × 104 GE) with phase I C. burnetii in phosphate-buffered saline (PBS). Uninfected controls were given PBS only. Mice were sacrificed 9 and 16 days postinfection.
Immunohistochemistry.The left pulmonary lobe, the right lateral liver lobe, and half the spleen from each infected mouse were harvested for histology. Tissues were fixed in 10% formalin overnight followed by 70% ethanol overnight and then processed and embedded in paraffin. Serial 5-μm sections were cut, deparaffinized with xylene, and rehydrated. Immunostaining was performed at room temperature. Cells on cytospins or paraffin-embedded tissue sections were incubated with 100 mM glycine for 20 min and then rinsed with PBS. Proteinase K (Dako, Carpinteria, CA) then was applied for 6 min. Samples were washed with Tris-buffered saline (TBS) and blocked with rodent block M (Biocare Medical, Concord, CA) for 30 min. They then were washed with TBS with 0.05% Tween 20 (TBS-T) before being incubated with polyclonal rabbit anti-C. burnetii at 1:8,000 in 0.5% TBS-T for 1 h at room temperature. After being washed again with TBS-T, a rabbit-on-rodent AP-polymer (Biocare Medical, Concord, CA) antibody was added for 30 min. Immunodetection was performed with Permanent Red (Dako, Carpinteria, CA) for 2 min and stopped with water. Tissues were counterstained with hematoxylin and coverslips mounted with aqueous Vectamount AQ mounting media (Vector Laboratories, Burlingame, CA). Images were acquired using a Nikon DS-Ri-1 camera mounted on a Nikon Eclipse 80i microscope, and the bacterial burden was determined by counting the number of bacterium-containing vacuoles per 20 random fields at ×20 or ×40 magnification that were verified to be within a cell wall by bright-field microscopy. Hematoxylin and eosin (H&E) staining was done by following the manufacturer's protocol. Pictures were taken, analyzed, and quantified by a blinded histotechnician.
Image analysis.Fiji by ImageJ was used to analyze fluorescent images. All images were acquired using a Nikon DS-Ri-1 camera mounted on a Nikon Eclipse 80i microscope and were 1,280 by 1,024 pixels using a 20× objective. Images were changed to 16-bit grayscale and threshold adjusted using intermodes. Binary images were created and fluorescent particles analyzed to achieve the percentage of the total area of the image that was positive for C. burnetii as revealed by antibody staining.
DNA extraction and PCR.One-half of the spleen and the remaining lobes of the lung not used for histology were homogenized and pelleted. Cellular pellets then were water lysed by vortex for 1 min and then centrifuged again at 500 × g for 5 min to remove cellular debris. Supernatant fluids containing C. burnetii were collected and centrifuged at 14,200 × g for 30 min to pellet the bacteria. The resulting pellet was resuspended in 1 ml sterile PBS, vortexed, and centrifuged at 500 × g for 5 min to remove more cellular debris. Supernatant fluids containing C. burnetii then were collected and centrifuged again at 14,200 × g for 30 min to pellet the bacteria. DNA extraction from the isolated bacteria was performed according to the manufacturer's protocol (UltraClean microbial DNA isolation kit; catalog number 12224; MoBio Laboratories, Inc.). Bacterial DNA was amplified in a quantitative real-time PCR with SYBR green PCR master mix (Applied Biosystems, Foster City, CA). Genomic equivalents were determined by amplification of rpoS gene copies as previously reported (28, 29). Reactions were performed with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). Results were compared to a standard curve generated using known amounts of C. burnetii DNA, and analyses were performed using Microsoft Excel and GraphPad Prism.
Chimera methods.Six-week-old recipient mice were lethally irradiated with 1,000 Gy administered in a split dose consisting of 500 Gy each with a 4-h rest between doses. Bone marrow cells (see below) were transferred via intravenous (i.v.) tail injection within 1 h of the second dose. The femur and tibia of donor mice were collected and flushed with 1 to 2 ml of ice-cold, sterile Dulbecco's PBS (DPBS) using a 26-guage needle and 3-ml syringe into a sterile petri dish. The marrow plug was disrupted to make a single-cell suspension by gently drawing it up and down in the needle. Cells from donor mice were pooled and filtered through 100-μm mesh cones into a 50-ml centrifuge tube. The bone marrow cells were washed in ice-cold DPBS and centrifuged at 1,000 to 1,200 rpm for 10 min. The supernatant fluid was decanted and the bone marrow cells were adjusted to approximately 5 × 107/ml. Two hundred microliters containing 1 × 107 cells was given i.v. to the irradiated mice. The mice were allowed to reconstitute for 8 to 9 weeks prior to challenge with C. burnetii, treated with antibiotic water for the first 6 weeks, and then given regular water until and during infection.
Statistical analysis.Statistical analyses were performed using Prism 4 (GraphPad Software, San Diego, CA). The data were analyzed by Student's t test or one- or two-way analysis of variance (ANOVA), as indicated. Two-way ANOVA was used when values were compared between and within groups, while one-way ANOVA was used when only comparing values between multiple groups.
RESULTS
Role of TLR2, TLR4, and MyD88 following peritoneal infection.Previous studies examining the roles of TLR2 and TLR4 in C. burnetii infection in vivo used the peritoneal route of infection and suggested a minimal role for these two receptors during C. burnetii infection (24, 26). A similar experiment was performed initially to confirm these observations and to ensure our method of analysis would yield similar results. Mice were infected i.p. with 1 × 104 GE, and disease course was monitored over time. Morbidity (weight loss) responses in infected TLR2−/− and TLR4−/− mice were similar to those that occurred in infected C57BL/6 controls, with TLR4−/− mice losing slightly less weight (Fig. 1A). When bacterial burden was measured by immunohistochemistry (IHC) 9 days postinfection, numbers of bacterium-infected cells in the spleen, heart, and liver were similar between TLR2−/− and C57BL/6 mice but actually were lower in the TLR4−/− mice (Fig. 1B). Overall, these initial studies support previous reports suggesting minimal protective roles for TLR2 and TLR4 following peritoneal C. burnetii infection.
Weight loss and bacterial counts after peritoneal C. burnetii infection. TLR2 and TLR4 do not promote C. burnetii clearance after peritoneal infection. C57BL/6, TLR2−/−, and TLR4−/− (n = 5 per group) mice were injected i.p. with 104 GE of phase I C. burnetii. (A) Mice were weighed daily, and results were recorded over 9 days. The graph represents average weights at each day postinfection, and error bars indicate standard errors of the means (SEM). Significance was determined by two-way ANOVA with Bonferroni's posttest. (B) C. burnetii burden in the heart, liver, and spleen was determined by immunohistochemistry. The graph represents pooled data, and error bars indicate SEM. Twenty images per slide were counted, and if total values exceeded 250, we were unable to distinguish differences with confidence. Therefore, when sample values were >250, we recorded them as such. Significance was determined by one-way ANOVA with Bonferroni's posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Roles for TLR2, TLR4, and MyD88 following pulmonary infection.Previous studies and our results described above suggest that TLR2 and TLR4 play a minimal role during peritoneal C. burnetii infection. However, C. burnetii is primarily a pulmonary pathogen, and these data may not accurately reflect the role of TLR2 and TLR4 in the natural route of infection. To examine the roles of TLR2 and TLR4 in pulmonary infection, C57BL/6, TLR2−/−, TLR4−/−, and MyD88−/− mice were infected i.t. with either 1 × 103 or 1 × 105 C. burnetii GE. C57BL/6, TLR2−/−, and TLR4−/− mice all exhibited outward morbidity, with weight loss beginning around day 4 at the high infectious dose and day 6 at the low infectious dose. MyD88−/− mice, however, showed no outward signs of morbidity and exhibited no weight loss over the entire course of infection (Fig. 2A).
Weight loss and tissue weights after pulmonary C. burnetii infection. C57BL/6, TLR2−/−, TLR4−/−, and MyD88−/− (n = 5 per group) mice were left uninfected or were infected i.t. with 103 or 105 GE of phase I C. burnetii. (A) Mice were weighed daily, and results were recorded over 9 days. (B) On day 9, mice were euthanized and spleens and lungs were removed and weighed. The graph represents pooled data normalized to body weight, and error bars indicate SEM. Significance was determined by two-way ANOVA with Bonferroni's posttest. P values were <0.05 (*) and <0.001 (***) compared to PBS for each mouse strain. P values were <0.01 (##) and <0.001 (###) compared to C57BL/6 mice for each infectious dose.
In addition to whole-body weights, changes in spleen and lung weights also were examined. For both infectious doses, the spleens and lungs of infected C57BL/6, TLR2−/−, and TLR4−/− mice weighed significantly more than uninfected tissues at 9 days postinfection. However, infected MyD88−/− spleens weighed more than control spleens only at the high infectious dose. Similarly, infected MyD88−/− lungs did not weigh significantly more than controls at either infectious dose, although infected MyD88−/− lungs did appear to trend toward increased weight at the high infectious dose (Fig. 2B). There were no differences in spleen weights between C57BL/6 and TLR4−/− mice. TLR2−/− mice had smaller spleens at the high infectious dose, and MyD88−/− mice had smaller spleens at both infectious doses (Fig. 2B). When lung weights were compared, there were no differences between C57BL/6, TLR2−/−, and TLR4−/− mice, but MyD88−/− mice had significantly smaller lungs at both infectious doses. The reduced weight gains in the MyD88-deficient tissues again correlated with reduced morbidity response in these animals. Thus, these gene deficiencies were associated with marked differences in pathological outcomes following lung infection with C. burnetii.
Analysis of tissues by histology showed necrosis in the livers of infected animals, particularly at the high dose of infection. C57BL/6, TLR2−/−, and TLR4−/− animals had similar amounts of liver necrosis. However, MyD88−/− animals had limited necrosis in their livers, further demonstrating reduced morbidity in these animals (Fig. 3). There was increased immune cell infiltration to the infected lungs in all mouse strains (Fig. 4). Interestingly, there were no distinguishable differences in immune cell infiltration between the mouse strains. Furthermore, when the recruitment of immune cells to the lung was analyzed by flow cytometry, similar numbers of recruited CD11b+ cells were observed between C57BL/6, TLR2−/−, TLR4−/−, and MyD88−/− mice (data not shown). Altogether, these results suggest that general inflammatory signals and immune cell recruitment following pulmonary C. burnetii infection did not critically depend on these molecules.
Liver damage after pulmonary C. burnetii infection. Representative histology pictures of C57BL/6, TLR2−/−, TLR4−/−, and MyD88−/− livers after i.t. infection with 105 GE of phase I C. burnetii. Arrows indicate sites of necrosis, which are absent from MyD88−/− livers. Images were taken at ×4 magnification.
Immune cell infiltration after pulmonary C. burnetii infection. Representative histology pictures of C57BL/6, TLR2−/−, TLR4−/−, and MyD88−/− lungs after i.t. infection with 105 GE of phase I C. burnetii. Arrows indicate recruited leukocytes observed around airways and blood vessels in all infected strains. Wild-type, TLR2−/−, and TLR4−/− infected tissues contain greater mononuclear cell infiltrate and loss of lacy architecture than MyD88−/− infected tissue. Images were taken at ×4 magnification.
To assess the amount of bacteria in spleens and lungs from infected mice at 9 days postinfection, bacterial numbers were analyzed by immunohistochemistry and blind counting of the number of infected vacuoles/20 fields/slide (IHC) and by PCR analysis of bacterial genomes (Fig. 5). In TLR2−/− mice, C. burnetii infection was greater in both the lung (as measured by IHC and PCR) and spleen (as measured by PCR) at day 9 than that for C57BL/6 mice. In TLR4−/− mice, bacterial burden was more prevalent in the spleens (as measured by IHC but not PCR) and lungs (as measured by IHC and PCR) of TLR4−/− mice than C57BL/6 mice at day 9. Finally, MyD88−/− mice exhibited an inability to effectively control the bacterial infection at either tissue site. Since some slides in the IHC analyses had too many positive vacuoles to confidently count (>250 total), the IHC stains also were analyzed by image analysis software, which showed the same trends, but the differences were not as dramatic (Fig. 5C). To ensure that these results were not specific to one time point, two separate additional experiments then were done in which the spleen and lung burdens of C. burnetii were further examined in TLR2−/− and TLR4−/− mice versus C57BL/6 controls at day 16 postinfection. Just as seen at day 9, higher C. burnetii burden was noted by PCR in spleens and lungs of TLR2-deficient mice versus controls following infection with either 1 × 103 or 1 × 105 genome equivalents at day 16 (Fig. 6). In contrast, no differences were noted in the spleens of TLR4-deficient animals at this time point, although higher bacterial burdens were seen in the lung, suggesting the trend noted at day 9 continued through day 16 (Fig. 7). Collectively, these results suggest a tissue-specific function for TLRs in pulmonary C. burnetii infection. Specifically, TLR2 and TLR4 appear to contribute to controlling bacterial growth and/or spread after pulmonary infection in mice, with TLR2 clearly having a more predominant role. Furthermore, while MyD88 signaling contributes to disease morbidity, it is also clearly essential for controlling C. burnetii growth and spread in both the lung and periphery.
C. burnetii burden after 9 days of pulmonary infection. C57BL/6, TLR2−/−, TLR4−/−, and MyD88−/− (n = 5 per group) mice were left uninfected or were infected i.t. with 103 or 105 GE of phase I C. burnetii. (A) Spleens and lungs were collected on day 9 postinfection, and C. burnetii-containing vacuoles were identified by immunohistochemistry and counted. Twenty images per slide were counted, and if total values exceeded 250, we were unable to distinguish differences with confidence. Therefore, when sample values were >250, we recorded them as such. (B) Spleens and lungs were collected on day 9 postinfection, and C. burnetii bacterial load was determined by PCR. (C) Image analysis of C. burnetii-stained area in immunohistochemistry images from mice infected i.t. with 103 GE. Three representative images were used from three separate experiments. Error bars indicate standard deviations (SD). Significance between groups was determined by two-way Student's t test compared to the corresponding wild type (WT). *, P < <0.05; **, P < 0.01; ***, P < 0.001.
C. burnetii burden in TLR2−/− mice after 16 days of pulmonary infection. C57BL/6 and TLR2−/− (n = 5 per group) mice were infected i.t. with 103 or 105 GE of phase I C. burnetii. Spleens and lungs were collected on day 16 postinfection, and the C. burnetii bacterial load was determined by PCR. Error bars indicate SD, and significance between groups was determined by two-way Student's t test compared to the corresponding wild type. *, P < 0.05; ***, P < 0.001.
C. burnetii burden in TLR4−/− mice after 16 days of pulmonary infection. C57BL/6 and TLR4−/− (n = 5 per group) mice were infected i.t. with 103 or 105 GE of phase I C. burnetii. Spleens and lungs were collected on day 16 postinfection, and the C. burnetii bacterial load was determined by PCR. Error bars indicate SD, and significance between groups was determined by two-way Student's t test compared to the corresponding wild type. *, P < 0.05.
TLR expression on bone marrow macrophages is likely of central importance in C. burnetii infection. However, TLRs are expressed on other cell types, including non-bone marrow-derived cells, such as epithelial cells (30). One of the potential differences between peripheral and pulmonary infection is the potential role of the lung epithelium. Therefore, bone marrow chimera mice were used to test the importance of TLR2 on bone marrow-derived versus non-bone marrow-derived cells in C. burnetii lung infection. TLR2 was chosen for these experiments, since TLR2−/− mice showed the most consistent phenotype in the experiments described above. Bone marrow chimera mice were generated in which TLR2-deficient bone marrow was established in irradiated wild-type mice and wild-type bone marrow was established in TLR2-deficient mice. Mice were infected with C. burnetii, and bacterial burdens in spleen and lung were compared at 9 days postinfection. As shown in Fig. 8, TLR2-deficient experimentally manipulated mice (TLR2-deficient bone marrow inserted into irradiated TLR2-deficient mice) had greater bacterial burden at day 9 in both spleen and lung than wild-type mice (wild-type bone marrow inserted into irradiated wild-type mice), confirming results discussed above. In chimera mice, greater bacterial burden was detected only in mice in which TLR2 was deficient in bone marrow-derived cells (Fig. 8), suggesting that TLR2 on hematopoietic cells is of central importance for the control of pulmonary C. burnetii infection.
C. burnetii burden in TLR2−/− chimera mice. Chimera mice were infected i.t. with 103 GE of phase I C. burnetii. Spleens and lungs were collected on day 9 postinfection, and the C. burnetii bacterial load was determined by PCR. Error bars indicate SD, and significance between groups was determined by two-way Student's t test compared to the corresponding wild type. *, P < 0.05.
DISCUSSION
Coxiella burnetii is the causative agent for Q fever in humans. Although this airborne pathogen can stimulate an efficient immune response that limits bacterial replication, 5 to 10% of hosts fail to completely clear the bacteria (31, 32). By mechanisms still not completely understood, C. burnetii can persist as a chronic infection by escaping the microbicidal activities of host cells, such as macrophages. Although the macrophage receptors TLR4 (26) and TLR2 (24) have been studied following C. burnetii infection in the peritoneum, their role in pulmonary infection, the natural route of infection, has not been examined. Our results demonstrate that the route of delivery of the bacterium impacts the roles for both TLR2 and TLR4 in the host response to C. burnetii. As such, a complete understanding of C. burnetii pathogenesis, particularly the roles of TLRs and other innate immune cell receptors, requires a more thorough analysis of the pulmonary disease.
In studies using a peritoneal infection model, investigators found that a lack of either TLR2 or TLR4 in mice did not result in significant differences in bacterial burden multiple days postinfection compared to that seen in control animals (24, 26). In our studies, we performed similar experiments and also found minimal consequences on morbidity and bacterial clearance in TLR2- and TLR4-deficient animals following peritoneal infection. In fact, clearance appeared to be enhanced in the absence of TLR4. However, when the same mice were used in a pulmonary infection model, significant and distinct roles for both TLRs in enhancing host defense in the target organ (lung) and/or dissemination to a distant organ (spleen) were seen at two different time points postinfection.
The morbidity response in C. burnetii-infected TLR2-, TLR4-, and MyD88-deficient animals provided unique insights into the pathology induced by the bacterium. Weight loss (cachexia) following infection was completely absent from MyD88-deficient animals, whereas it still occurred in the TLR2- or TLR4-deficient mice. In addition to cachexia, similar patterns were observed for liver necrosis. Thus, the MyD88-dependent inflammatory cascade leading to the production of inflammatory mediators, such as tumor necrosis factor alpha (TNF-α) (cachexin), most likely accounts for this observation. Interestingly, the absence of a significant reduction in cachexia or liver necrosis in TLR2- and TLR4-deficient animals suggests that other MyD88-dependent receptors, such as other TLRs or interleukin-1 (IL-1) family receptors, contribute to the cachexia and liver necrosis. The use of additional gene deletion animals for these other receptors in the same infection model should be useful for determining which pathways are critical for C. burnetii-induced morbidity.
Surprisingly, when general inflammatory responses were studied in the lungs from infected TLR2-, TLR4-, and MyD88-deficient animals, we found that enhanced cellular recruitment of inflammatory cells occurred in each of the deficient animals. Therefore, inflammatory pathways were triggered in these mice, but because they lacked the MyD88 contribution, these responses were not protective. Large numbers of neutrophils were seen in infected tissues from the deficient and control animals, yet there were significant differences in bacterial burden, suggesting these innate cells have minimal effects on clearance of the bacterium, as suggested by others (33, 34).
When bacterial burden was examined, TLR2-deficient mice had a greater bacterial burden in the lungs than control mice following pulmonary infection at 9 and 16 days postinfection. Furthermore, our chimera studies showed that only mice transplanted with TLR2-deficient bone marrow cells had greater bacterial burden than control mice. These data suggest that TLR2 expression on hematopoietic cells has an important role in preventing bacterial growth at the site of infection in the lung. Signaling through TLR2 by phase II C. burnetii is known to promote IL-12 production (23), which could lead to the production of gamma interferon (IFN-γ), an important cytokine in efficient host immunity against C. burnetii (35). However, some reports suggest that phase I C. burnetii does not promote inflammatory TLR2 signaling, which was supported by data that demonstrate little role for TLR2 in bacterial clearance in the spleen or liver after peritoneal infection (24). However, Meghari et al. also showed that TLR2 is being triggered during these infections, as granuloma development in the spleen and liver was reduced in TLR2−/− mice (24). In contrast to these peritoneal infection studies, we found that bacterial clearance in the spleen was reduced in TLR2−/− mice after pulmonary infection, as measured by PCR but not IHC. This difference likely is due to the fact that IHC measures Coxiella-containing vacuoles but does not determine how many bacteria are contained within each vacuole. These data suggest that while the absence of TLR2 did not lead to an increased number of infected macrophages in the spleen, the ability of these TLR2−/− macrophages to control intracellular bacterial numbers was reduced. In support of this, TLR2-deficient macrophages have been found to not be as effective at controlling the establishment of infection in vitro (23). We observed similar results with in vitro infection of peritoneal and pulmonary macrophages (data not shown). Also, recently it has been shown that TLR1/TLR2 in human cells can be an important recognition receptor for C. burnetii infection, leading to robust cytokine responses (36). Therefore, the absence of TLR2 signaling may allow for the development of permissive populations of macrophages for C. burnetii growth. However, results of peritoneal infection studies suggest that these TLR2-mediated responses are more important in the lung environment than they are in the periphery.
In contrast to results for TLR2-deficient mice, IHC data suggest TLR4-deficient mice had increased numbers of infected cells in the spleens at day 9 after pulmonary infection. Furthermore, although PCR did not show an increased splenic burden, the data did show increased bacterial burden in the lungs at days 9 and 16. These data suggest that TLR4 does contribute to minimizing the spread of the bacteria in the lung and spleen. One way TLR4 may contribute to this is by reducing the establishment of permissive macrophages. However, previous reports (23) and our preliminary experiments do not observe increased bacterial loads in pulmonary and peripheral macrophages from TLR4−/− mice after in vitro infection (data not shown). Therefore, a direct increase in the number of permissive macrophages likely does not explain changes in bacterial loads between control and TLR4−/− mice. As an alternative mechanism, TLR4 signaling has been shown to promote the production of IFN-γ and TNF-α by murine splenocytes in response to C. burnetii (26), which could enhance the anti-Coxiella responses of infected macrophages indirectly. However, our data demonstrating that TLR4 was actually detrimental after peripheral infection in the spleen suggest that this is not sufficient to explain our results after pulmonary infection. Rather, these findings suggest that TLR4 has a unique lung-specific role. TLR4, like TLR2, was shown previously to promote the development of granulomas after peritoneal C. burnetii infection, although this did not seem to influence bacterial growth and clearance in these experiments (26). However, it is possible that this granuloma response is useful for preventing the spread of C. burnetii during pulmonary infection. Future studies should examine granuloma formation in the lung after pulmonary C. burnetii infection and their role in containing C. burnetii within the lung.
Finally, when MyD88-deficient mice were infected, they showed a more dramatic reduction in clearance in both lung and spleen. Thus, while MyD88 signaling is absolutely critical for early cachexia and liver necrosis during pulmonary C. burnetii infection, it is also critical for controlling bacterial growth and spread. Interestingly, the overall phenotype in MyD88-deficient mice is greater than the combined phenotype of TLR2−/− and TLR4−/− mice. These data suggest that other TLRs, such as TLR9, and/or members of the IL-1 receptor family, such as IL-1R, IL-18R, and/or IL-33R, are involved in activating the anti-Coxiella response by macrophages and promoting host defense against C. burnetii. Our conclusions concerning the role for MyD88-mediated inflammation in controlling the dissemination of C. burnetii from the lung are similar to conclusions by Calverley et al., who also found that the host inflammatory response is important in controlling dissemination of C. burnetii from the lung (37).
In conclusion, our experiments clearly show that TLR2 and TLR4 are critical for optimal host defense during pulmonary C. burnetii infection and that this bacterium does not completely avoid TLR signaling. Thus, the prevailing view that TLR2 and TLR4 responses are not important against this bacterium for preventing bacterial growth should be revisited. Our results are consistent with the recent demonstration of TLR1/2 recognition of C. burnetii in human cells (36), which is counter to the earlier view that these TLR2 ligands are masked (25). Finally, our studies illustrate the importance of conducting experiments using relevant routes and tissues for infection to fully appreciate the role of key innate immune cell recognition receptors in pathogenesis. Such information is important in developing and testing new countermeasures for use in treating Q fever in humans.
ACKNOWLEDGMENTS
We thank Robert Heinzen (Rocky Mountain Laboratories, NIH/NIAID, Hamilton, MT) for the kind donation of C. burnetii Nine Mile phase I (RSA493).
This work was supported by NIH grant 1 R21 AI094261, National Institutes of Health IDeA Program grant GM110732, an equipment grant from the M. J. Murdock Charitable Trust, and The Montana State University Agricultural Experimental Station.
FOOTNOTES
- Received 13 July 2015.
- Returned for modification 3 August 2015.
- Accepted 14 January 2016.
- Accepted manuscript posted online 19 January 2016.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
