Neutrophils Play an Important Role in Protective Immunity against Coxiella burnetii Infection

Abstract

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes the zoonotic disease Q fever. Although Q fever is mainly transmitted by aerosol infection, study of the immune responses in the lung following pulmonary C. burnetii infection is lacking. Neutrophils are considered the first immune cell to migrate into the lung and play an important role in host defense against aerosol infection with microbial pathogens. However, the role of neutrophils in the host defense against C. burnetii infection remains unclear. To determine the role of neutrophils in protective immunity against C. burnetii infection, the RB6-8C5 antibody was used to deplete neutrophils in mice before intranasal infection with C. burnetii. The results indicated that neutrophil-depleted mice developed more severe disease than their wild-type counterparts, suggesting that neutrophils play an important role in host defense against C. burnetii pulmonary infection. We also found that neither CXC chemokine receptor 2 (CXCR2) nor interleukin-17 (IL-17) receptor (IL-17R) deficiency changed the severity of disease following intranasal C. burnetii challenge, suggesting that keratinocyte-derived chemokine and IL-17 may not play essential roles in the response to C. burnetii infection. However, significantly higher C. burnetii genome copy numbers were detected in the lungs of IL-1R^−/− mice at 14 days postinfection. This indicates that IL-1 may be important for the clearance of C. burnetii from the lungs following intranasal infection. Our results also suggest that neutrophils are involved in protecting vaccinated mice from C. burnetii challenge-induced disease. This is the first study to demonstrate an important role for neutrophils in protective immunity against C. burnetii infection.

INTRODUCTION

Coxiella burnetii is an obligate intracellular bacterium that causes acute and chronic Q fever in humans. The infection is mainly transmitted through inhalation of C. burnetii-contaminated materials, with the infectious dose in humans being as low as 1 to 10 organisms (1). Once inside the host cell, C. burnetii replicates within a highly acidic parasitophorous vacuole (PV) which shares markers with a secondary lysosome (2). Because the organism is highly resistant to environmental stresses, such as UV radiation and drying, and because of its ability to be spread through aerosol, its high infectivity, and the severity of disease with chronic infection, it is considered a category B select agent.

The first cells which a pathogen encounters when entering the lung are alveolar epithelial cells and alveolar macrophages. Macrophages and epithelial cells recognize bacteria through the binding of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) on and within the cell to pathogen-associated molecular patterns (PAMPs) on the bacteria. The activation of these receptors leads to the release of inflammatory cytokines, such as interleukin-1β (IL-1β), IL-8, and macrophage inflammatory protein 1 (MIP-1), which cause localized inflammation, including the infiltration of neutrophils within 24 h postinfection (p.i.) (3). However, a previous study from our lab found that following C. burnetii aerosol infection, neutrophils are not present in the airways until 7 days p.i. (4). The mechanism of this delay is unknown.

CXC chemokine receptor 2 (CXCR2) is the major receptor regulating inflammatory neutrophil recruitment in inflamed tissues. In mice, keratinocyte-derived chemokine (KC) is released at the site of infection by macrophages and epithelial cells and binds to CXCR2 on the surface of the neutrophil. This causes the neutrophil to upregulate adhesion molecules, such as selectins and integrins, which allow circulating neutrophils to slow down and attach to the vascular endothelium (5). Neutrophils follow a gradient of increasing amounts of KC and other chemoattractants to travel toward the site of infection. CXCR2-knockout mice have decreased neutrophil recruitment, an increased bacterial burden in the lungs, and increased mortality following intratracheal challenge with Streptococcus pneumoniae (6). The role of CXCR2 following intranasal infection with C. burnetii has not been studied.

After migrating to the site of infection, neutrophils engulf and destroy bacteria. They contain highly bactericidal molecules within their granules, such as myeloperoxidase and lysozyme, and produce highly toxic reactive oxygen species (ROS), such as H₂O₂ (7). Once a neutrophil engulfs a bacterium, the neutrophils produce inflammatory cytokines to promote the migration of more cells toward the site of infection and increase cell proliferation. Previous studies have shown the importance of alveolar neutrophils through the delayed clearance of S. pneumoniae, Legionella pneumophila, and Klebsiella pneumoniae when neutrophils were selectively depleted (8–10). However, the role that neutrophils play in the host defense against C. burnetii infection has not been studied in depth.

Formalin-inactivated C. burnetii Nine Mile phase I (NMI) whole-cell vaccine (phase I vaccine [PIV]) has been found to induce long-lasting protective immunity against challenge with virulent C. burnetii NMI (11). Our recent study demonstrated that the passive transfer of immune serum from PIV-vaccinated CD4⁺ T-cell-deficient mice conferred significant protection against C. burnetii challenge in naive recipient mice (12). Furthermore, purified IgM from PIV-vaccinated CD4⁺ T-cell-deficient mouse serum inhibited C. burnetii infection in mice, suggesting that T-cell-independent anti-phase I-specific IgM may play a critical role in PIV-induced protection against C. burnetii infection (12). A recent study (13) found in the spleen a specific group of neutrophils which induce T-cell-independent IgM production by marginal zone (MZ) B cells. These neutrophils, named “B-cell-helper neutrophils,” activate MZ B cells equally as effectively as they activate splenic CD4⁺ T cells but are more effective than macrophages and dendritic cells. However, it is unknown whether neutrophils play a role in activating B cells to generate protective T-cell-independent anti-PIV-specific IgM and whether neutrophils contribute to PIV-induced protective immunity against C. burnetii infection.

In the current study, to understand the role of neutrophils in protective immunity against C. burnetii infection, we examined if depletion of neutrophils in mice would significantly affect the ability of PIV to confer protection against C. burnetii pulmonary infection. The results indicate that neutrophils are required for both the host immune response to primary infection and vaccine-induced protection against C. burnetii. This is the first study to demonstrate that neutrophils play an important role in protective immunity against C. burnetii infection.

MATERIALS AND METHODS

Animals.

Specific-pathogen-free 8-week-old female BALB/c, B6, CXCR2^−/−, IL-17R^−/−, and IL-1R^−/− mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in sterile microisolator cages under specific-pathogen-free conditions at the University of Missouri (MU) laboratory animal facility. Following intranasal infection with C. burnetii NMI, the mice were housed in an animal biosafety level 3 (ABSL3) facility at the University of Missouri Laboratory for Infectious Disease Research (MU-LIDR). All research protocols described in this study were approved by the Institutional Biosafety Committee and the Animal Care and Use Committee of the University of Missouri.

Bacterial strain.

C. burnetii NMI clone 7 (RSA493) was used for both in vitro and in vivo experiments. The bacteria were propagated in L929 cells and purified by density centrifugation as previously described (14). NMI was handled under biosafety level 3 (BSL3) conditions in the MU-LIDR.

Neutrophil depletion.

Antibody RB6-8C5-producing hybridoma cells were a kind gift from Deb Anderson's lab at the University of Missouri. The RB6-8C5 monoclonal antibody binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils and small subpopulations of inflammatory monocytes, T cells, and dendritic cells (15). Hybridoma cells were grown in hybridoma serum-free medium (Gibco, Grand Island, NY), and the culture supernatant was collected every 3 days. The supernatant was then run through a high-affinity protein G agarose column (Pierce) to purify the RB6-8C5 antibodies. The antibody concentration was determined using a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL). To deplete neutrophils in mice, 0.25 mg of RB6-8C5 antibody in 100 μl of phosphate-buffered saline (PBS) was injected intraperitoneally into each mouse 1 day prior to infection and then every other day until sacrifice. Preliminary experiments showed that RB6-8C5 injection decreased the amount of circulating neutrophils to less than 5% of the cell population. RB6-8C5 injection had no deleterious effect on uninfected mice.

Animal infection.

Mice were anesthetized using isoflurane gas. A total of 1 × 10⁷ C. burnetii NMI bacteria in 30 μl of PBS was used to challenge each mouse via the intranasal route. Mice were positioned with their nose pointed upwards, and a pipette was used to deliver the fluid into the nostrils during inhalation.

BALF collection.

Bronchoalveolar fluid (BALF) was collected as described previously, with modifications (16). Following euthanasia of the mice, the trachea was exposed and a 22-gauge catheter was inserted and secured with suture thread. To obtain BALF, 1 ml of Hanks balanced salt solution (HBSS; HyClone Labs, Logan, UT) was slowly infused into the lungs and then withdrawn. This procedure was repeated 3 times. The fluid was centrifuged at 300 × g for 5 min, and the supernatant was collected and frozen at −80°C. Cells were spun onto a glass slide using a cytocentrifuge and stained with Diff-Quick to determine the cell population.

Isolation of neutrophils and macrophages.

Neutrophils were collected from the bone marrow of the mice as described previously (17). The mice were euthanized, the femurs and tibias were removed, and the ends of the femurs and tibias were cut off, exposing the marrow. A 25-guage needle was placed into the end of the bone, and 3 ml of HBSS was flushed through the bone. Red blood cells were lysed by incubating the marrow with ACK buffer (Lonzana, Walkersville, MD) for 5 min. Mononuclear cells were separated from the neutrophils using discontinuous density centrifugation with 55%, 65%, and 75% Percoll solutions (GE Healthcare, Pittsburg, PA). Three milliliters of a 75% solution of Percoll in PBS was layered under 3 ml of a 65% solution, and then 3 ml of a 55% solution was added on top of the 65% solution. The marrow cells suspended in 3 ml HBSS were layered on top of the 55% solution, and the tube was centrifuged at 500 × g for 30 min. The neutrophils isolated from between the 65% and 75% layers were counted and processed for further experiments, described later. Trypan blue was used to assess cell viability, which was greater than 98% for each assay.

Neutrophil infection.

A total of 1 × 10⁵ freshly isolated neutrophils was placed into each well of a 24-well plate containing RPMI and 10% fetal bovine serum (FBS) at 37°C. C. burnetii NMI was added to the neutrophils at a multiplicity of infection (MOI) of 100 for different times. Escherichia coli lipopolysaccharide (LPS) was added at a concentration of 10 μg/ml for use as a control. The culture supernatant was collected and frozen at −80°C for further analysis.

PIV vaccination.

Purified C. burnetii NMI was inactivated by the use of a 1% formaldehyde solution as described previously (18). The protein concentration of inactivated PIV antigen was measured by using a micro-BCA protein assay kit (Pierce, Rockford, IL). Mice were vaccinated through the subcutaneous injection of 4 μg of PIV in 50 μl PBS plus 50 μl aluminum hydroxide (Sigma).

Real-time PCR.

Lung and spleen tissue was lysed with 200 μl lysis buffer (1 M Tris, 0.5 M EDTA, 7 mg/ml glucose, 28 mg/ml lysozyme) and 10 μl proteinase K (20 mg/ml) and incubated for 18 h at 60°C. Following the addition of 21 μl 10% sodium dodecyl sulfate (SDS), samples were incubated at room temperature for 1 h. DNA was extracted using a High Pure PCR template preparation kit (Roche Molecular Biomedicals, Indianapolis, IN) and stored at −80°C until use. Real-time PCR (rtPCR) was performed using an Applied Biosystems 7300/7500 real-time PCR system. Recombinant plasmid DNA (consisting of the com1 gene ligated into the PET23a vector) was used as standard DNA to quantify the com1 gene copy numbers for assessment of the bacterial burden.

Histopathology.

Lungs and spleens were removed from the mice at different time points and fixed in 10% formalin for at least 72 h. Tissues were sectioned, embedded in paraffin, and cut to a thickness of 5 μm. They were allowed to adhere to glass slides and stained with hematoxylin and eosin. The slides were viewed and scored by a trained pathologist.

ELISA.

Proinflammatory cytokines MIP-1, KC, tumor necrosis factor alpha (TNF-α), and IL-1β were detected from the BALF of infected mice and the culture supernatants of infected cells using a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (eBioscience, San Diego, CA).

Statistical analysis.

The results, expressed as means ± standard deviations, were compared by Student's t test. Differences were considered significant at a P value of ≤0.05.

RESULTS

Neutrophils play a critical role in host defense against primary C. burnetii infection.

To understand what role neutrophils play in host defense against C. burnetii pulmonary infection, we examined if neutrophil deficiency in mice would significantly increase their susceptibility to C. burnetii intranasal infection. Depletion of polymorphonuclear leukocytes (PMNs) was performed by treating mice with the monoclonal antibody RB6-8C5. The efficiency of neutrophil depletion was confirmed by preliminary experiments which showed that RB6-8C5 injection decreased the amount of circulating neutrophils to less than 5% of the cell population. The severity of the disease induced by the C. burnetii intranasal infection in PMN-depleted and IgG-treated control mice was evaluated by comparing body weight loss, the level of splenomegaly, the bacterial burden, and pathological changes in the lung and spleen at different time points postinfection (p.i.). As shown in Fig. 1A, both PMN-depleted and IgG-treated control mice had a significant body weight loss at 8 days p.i. However, the control mice recovered more quickly, returning to their day 0 body weight by day 13, while PMN-depleted mice did not return to their day 0 body weight until day 18 p.i. PMN-depleted mice also had a significantly lower body weight than the control mice on both day 11 and day 13 p.i. These observations suggest that neutrophils may play an important role in protecting the host from developing clinical disease. As shown in Fig. 1B, the level of splenomegaly was significantly increased on days 7, 14, and 28 days p.i. in PMN-depleted mice compared to that in control mice. Additionally, significantly higher C. burnetii genome copy numbers were also detected in the spleens from PMN-depleted mice than in the spleens from the control mice on days 14, 28, and 40 days p.i. (Fig. 1C). These results suggest that neutrophils play a critical role in bacterial clearance from the spleen in both the early and late stages of C. burnetii infection. In addition, significantly higher C. burnetii genome copy numbers were detected in the lungs from PMN-depleted mice than in the lungs from control mice on day 28 p.i. (Fig. 1D). Collectively, the increased disease severity observed in PMN-depleted mice occurs mainly with early infection, peaking at 14 days p.i., but continues for as long as 40 days p.i., suggesting that neutrophils play an important role in controlling local and systemic infection. Furthermore, to understand the inflammatory response in the lung and spleen tissues following pulmonary infection, the histopathological changes in the lungs and spleens from PMN-depleted and control mice were examined and scored. The histology samples from the lungs of infected mice revealed increased inflammation on days 7 and 14 p.i. but no difference in inflammation between PMN-depleted and control mice (data not shown). However, histology samples from the spleens of infected mice revealed a higher number of macrophages in PMN-depleted mice than control mice at day 14 p.i. (Fig. 1E). The histology score for both groups did not increase until day 14 p.i. and was significantly higher for PMN-depleted mice at 14 day p.i. (Fig. 1F). These results suggest that neutrophils also play an important role in regulating the host inflammatory response against C. burnetii systemic infection.

FIG 1.

Neutrophil-depleted mice have increased disease severity following C. burnetii infection. BALB/c mice were injected with RB6-8C5 antibody (to deplete PMNs) or IgG antibody (as a control) 1 day prior to and throughout the infection. Mice were then challenged with 1 × 10⁷ C. burnetii NMI bacteria intranasally and sacrificed on days 3, 7, 14, 28, and 40 p.i. (A) The relative body weight (current body weight/day 0 body weight) was collected throughout the infection. (B) During necropsy, spleens were weighed and the level of splenomegaly was determined from the ratio of the spleen weight to the body weight. (C) DNA was collected from the spleen, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. (D) DNA was collected from the lung, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. (E) Spleens were fixed, embedded in paraffin, and stained with hematoxylin and eosin. A large accumulation of macrophages can be seen in PMN-depleted mice at day 14 p.i. (outlined in black). (F) Tissue sections were scored by a trained pathologist and were assigned the following scores: 0, which indicates no accumulation of macrophages; 1, which indicates a few small accumulations of macrophages; 2, which indicates a few small to moderate accumulations of macrophages; and 3, which indicates large numbers of moderate to large accumulations of macrophages. *, P ≤ 0.05 (n = 5 mice per group).

Cytokines within the lungs following C. burnetii infection.

To examine the cytokine responses in the lung tissue following intranasal infection, BALF was collected from infected mice and used to determine cytokine levels in neutrophil-depleted and control mice at early and late time points. As shown in Fig. 2A, the level of KC, which is a major neutrophil chemoattractant following infection, was significantly lower in PMN-depleted mice than control mice at day 7 p.i. However, although the level of KC decreased in both groups at day 14 p.i., PMN-depleted mice had significantly higher levels of KC than control mice. Contrary to the findings for the KC levels in the lungs, the level of MIP-1, which is also a chemoattractant for neutrophils, was significantly increased at day 7 p.i. in PMN-depleted mice (Fig. 2B). The amount of MIP-1 decreased to similar levels in both groups by day 14 p.i. The concentration of TNF-α, a proinflammatory cytokine, was similar in the two groups on day 7 p.i. (Fig. 2C). However, similar to the findings for KC, PMN-depleted mice had a significantly larger amount of TNF-α than control mice by day 14. The increased levels of KC and TNF-α in PMN-depleted mice at day 14 p.i. suggest that the lung inflammation is not resolved in PMN-depleted mice as soon as it is in control mice. All cytokines were at similar levels by day 28 p.i. Collectively, these results suggest that PMN depletion at the time of infection leads to differences in early cytokine responses within the lungs. These differences are likely tied to the increased disease severity observed in PMN-depleted mice following infection with C. burnetii.

FIG 2.

Cytokines within the lungs following C. burnetii infection. BALB/c mice were injected with RB6-8C5 antibody (to deplete PMNs) or IgG antibody (as a control) 1 day prior to and throughout the infection. BALF samples were collected from mice infected intranasally with 1 × 10⁷ C. burnetii NMI bacteria. The level of KC (A), the amount of MIP-1 (B), and the concentration of TNF-α (C) were determined using an ELISA kit. *, P ≤ 0.05 (n = 5 mice per group).

Cytokine levels of C. burnetii-infected neutrophils in vitro.

There are many different cell types within the lung during a bacterial infection. While macrophages and epithelial cells are the first to initiate inflammation, neutrophils encountering bacteria can produce cytokines to increase inflammation. To determine whether neutrophils contribute to the production of proinflammatory cytokines during C. burnetii infection, we measured the concentrations of IL-1β and TNF-α in the culture supernatant of C. burnetii NMI-infected neutrophils at different time points. As shown in Fig. 3A, compared to the level of IL-1β in the negative control, a low level of IL-1β was detected from C. burnetii-infected neutrophils at both 1 h and 24 h p.i., but the concentration at 1 h p.i. was higher than that at 24 h p.i., suggesting that IL-1β may be a cytokine released from neutrophils early in response to C. burnetii infection. In contrast, a high level of TNF-α was detected from C. burnetii-infected neutrophils only at 24 h p.i., suggesting that TNF-α may be a cytokine released from neutrophils at a later time in response to C. burnetii infection (Fig. 3B). These results suggest that neutrophils may also play an important role in releasing proinflammatory cytokines to induce early inflammatory responses against C. burnetii infection.

FIG 3.

Cytokines produced by neutrophils during C. burnetii infection. Neutrophils were collected from bone marrow and infected with C. burnetii NMI at an MOI of 100. E. coli LPS was added at a concentration of 10 μg/ml for use as a control. Medium was collected at 1 h and 24 h following infection. A commercially available ELISA kit was used to determine the concentrations of IL-1β (A) and TNF-α (B) in the medium. Data are for 5 mice per group. Dashed lines, limits of detection.

The neutrophil depletion phenotype is CXCR2 independent.

Because cytokine analysis of the BALF from infected mice showed differences in cytokine levels between PMN-depleted and control mice, the next step was to determine which pathway is involved in immune defense following infection. CXCR2 is the major receptor regulating inflammatory neutrophil recruitment in inflamed tissues and serves as the receptor for KC. PMN-depleted mice had a significantly higher level of KC than control mice, suggesting that the CXCR2 pathway may play a major role in recruiting neutrophils in response C. burnetii pulmonary infection. To test this hypothesis, we examined if CXCR2 deficiency in mice would significantly increase their susceptibility to C. burnetii intranasal infection. As shown in Fig. 4A, the level of splenomegaly was significantly increased in PMN-depleted mice but was similar between wild-type (WT) and CXCR2 ^−/− mice. Similar to the findings for splenomegaly, the bacterial burden was significantly increased in PMN-depleted mice but similar between WT and CXCR2^−/− mice (Fig. 4B). However, the bacterial burden in the lungs was not significantly different among the groups (Fig. 4C). These observations suggest that the CXCR2 pathway may not play a major role in recruiting neutrophils in response to C. burnetii pulmonary infection.

FIG 4.

CXCR2 is not involved in the neutrophil response to C. burnetii infection. BALB/c mice, CXCR2^−/− mice, or mice depleted of PMNs by the use of monoclonal antibody RB6-8C5 were infected intranasally with 1 × 10⁷ C. burnetii NMI bacteria. Mice were sacrificed on day 14 p.i. (A) The spleens were weighed, and the level of splenomegaly was determined using a ratio of the spleen weight to the body weight. (B) DNA was collected from the spleen, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. (C) DNA was collected from the lung, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. *, P ≤ 0.05 (n = 5 mice per group).

Role of IL-1 and IL-17 following C. burnetii pulmonary infection.

To determine whether IL-1 and IL-17 are involved in the pulmonary immune response to C. burnetii infection, we examined if mice lacking the IL-1 receptor (IL-1R) or the IL-17 receptor (IL-17R) had significantly different immune responses against intranasal infection. As shown in Fig. 5, the level of splenomegaly and the bacterial burden in the spleen and lung were similar between IL-17R^−/− and WT mice, suggesting that IL-17 may not play an essential role in response to C. burnetii intranasal infection. In contrast, although there was no significant difference in the level of splenomegaly and the bacterial burden in the spleen between IL-1R^−/− and WT mice at different time points postinfection, significantly higher C. burnetii genome copy numbers were detected in the lungs from IL-1R^−/− mice than in those from WT mice at 14 days p.i. These results suggest that IL-1 may not be involved in recruiting neutrophils but plays an important role in the clearance of C. burnetii from the lungs following intranasal infection.

FIG 5.

Role of IL-17 and IL-1 following C. burnetii infection. B6 mice, IL-17R^−/− mice, and IL-1R^−/− mice were infected intranasally with 1 × 10⁷ C. burnetii NMI bacteria. Mice were sacrificed on days 7, 14, and 30 p.i. (A) The spleens were weighed, and the level of splenomegaly was determined using a ratio of the spleen weight to the body weight. (B) DNA was collected from the spleen, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. (C) DNA was collected from the lung, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. (*, P ≤ 0.05; n = 5 mice per group).

Neutrophils are involved in PIV-induced protection.

Our recent study (12) suggests that T-cell-independent anti-phase I-specific IgM plays a critical role in PIV-induced protection against C. burnetii infection. To determine if neutrophils play a role in vaccine protection, we investigated if depletion of neutrophils in mice during vaccination with PIV would significantly affect the ability of PIV to elicit protective immunity against C. burnetii infection. Mice were injected with neutrophil-depleting antibody RB6-8C5 or control IgG 1 day prior to and every other day after vaccination with PIV. To replenish the neutrophil population before challenge with C. burnetii, depletion of neutrophils was discontinued 1 week prior to challenge. A previous pilot study found that neutrophil levels in the blood are restored within 4 days of the discontinuation of the RB6-8C5 injections. PIV-vaccinated neutrophil-depleted and control mice were challenged with C. burnetii NMI via the intranasal route at 28 days postvaccination. The protective efficacy of PIV in PMN-depleted and control mice was evaluated by comparing body weight loss, the level of splenomegaly, and the bacterial burden in the lung and spleen at 14 days postchallenge. The results indicated that there was no significant difference in body weight loss, the level of splenomegaly, or the bacterial burden in the lung between PMN-depleted and control mice (Fig. 6), suggesting that neutrophils may not play a critical role in PIV-induced protective immunity during vaccination. In addition, to determine if neutrophils play a role in PIV-induced protective immunity during C. burnetii challenge, we examined if the depletion of neutrophils in PIV-vaccinated mice during C. burnetii challenge would significantly affect the ability of PIV to confer protection against C. burnetii infection. PIV-vaccinated mice were treated with neutrophil-depleting antibody RB6-8C5 or the IgG control 1 day prior to and every other day after intranasal infection with C. burnetii. As shown in Fig. 7A, PMN-depleted mice had a significantly lower relative body weight than control mice. In addition, the level of splenomegaly was also significantly higher in PMN-depleted mice than in IgG-treated control mice (Fig. 7B). These observations suggest that neutrophils may play an important role in protecting PIV-vaccinated mice from the severe clinical disease induced by C. burnetii intranasal infection. Interestingly, there was no significant difference in bacterial burdens in the lungs and spleen between PIV-vaccinated PMN-depleted and control mice (Fig. 7C). This result suggests that neutrophils may not play an important role in the increased bacterial clearance observed in vaccinated mice following infection. Overall, these observations suggest that neutrophils may play an important role in protecting PIV-vaccinated mice from C. burnetii intranasal challenge-induced disease.

FIG 6.

Neutrophils do not play a role in PIV protection against C. burnetii infection at the time of vaccination. BALB/c mice were injected with RB6-8C5 antibody (to deplete PMNs) or IgG antibody (as a control) 1 day prior to and throughout vaccination with PIV. RB6-8C5 antibody was discontinued 1 week prior to infection to allow the neutrophil population to reestablish itself. Mice were challenged intranasally with 1 × 10⁷ C. burnetii NMI bacteria and were sacrificed on day 14 p.i. (A) Relative body weight (current body weight/day 0 body weight) was recorded throughout the infection. (B) The spleens were weighed, and the level of splenomegaly was determined using a ratio of the spleen weight to the body weight. (C) DNA was collected from the lung, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. Data are for 5 mice per group.

FIG 7.

Neutrophils play a role in PIV protection against C. burnetii infection at the time of infection. BALB/c mice were vaccinated with formalin-killed PIV. At 28 days after vaccination, mice were injected with RB6-8C5 antibody (to deplete PMNs) or IgG antibody (as a control) 1 day prior to and throughout the infection. Mice were challenged intranasally with 1 × 10⁷ C. burnetii NMI bacteria and were sacrificed on day 14 p.i. (A) Relative body weight (current body weight/day 0 body weight) was recorded throughout the infection. (B) The spleens were weighed, and the level of splenomegaly was determined using a ratio of the spleen weight to the body weight. (C) DNA was collected from the spleen and lung, and rtPCR was used to determine the genomic copy number of C. burnetii NMI. *, P ≤ 0.05 (n = 5 mice per group).

DISCUSSION

When bacterial pathogens enter the lungs, neutrophils are usually the first immune cells to accumulate at the site of infection and are very important for clearance of the bacteria from the lungs. However, it remains unclear what role neutrophils play in host defense against C. burnetii pulmonary infection. To fill the gap in the knowledge of the pulmonary immune response against C. burnetii infection, the current study focused on examining the role of neutrophils following intranasal infection with C. burnetii. The observation that C. burnetii intranasal infection induced more severe disease in PMN-depleted mice than in control mice provided strong evidence to support the suggestion that neutrophils play an important role in host defense against C. burnetii pulmonary infection. In addition, the current study also provided interesting information to help provide an understanding of what occurs when bacteria enter the airway, including the production of cytokines and chemokines, which increase inflammation and activate neutrophils. To our knowledge, this is the first study that has attempted to obtain an understanding of the mechanisms of neutrophil-mediated protective immunity against C. burnetii pulmonary infection.

Pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), are expressed on and inside alveolar macrophages and alveolar epithelial cells (19). The activation of PRRs leads to the production of proinflammatory cytokines, such as IL-1β and TNF-α, as well as chemokines, such as IL-8 or KC, in mice. Previous studies have shown that TNF-α is one of the proinflammatory cytokines which is produced by human and murine macrophages during C. burnetii infection (20, 21). Macrophages from mice deficient in TLR2 and TLR4 have decreased amounts of TNF-α production following C. burnetii infection, suggesting a role for these two receptors in macrophage activation and inflammation (22, 23). In the current study, TNF-α was at the highest levels in the lungs at day 7 p.i. in both PMN-depleted and control mice, suggesting that immune cells were able to recognize C. burnetii without the presence of neutrophils. On day 14, TNF-α levels decreased considerably; however, the PMN-depleted mice had a larger amount than the control mice. This, along with the observation that there was a slightly larger amount of C. burnetii in the lungs in PMN-depleted mice on day 14, suggests that neutrophils could play a role in the clearance of C. burnetii from the lungs. TLR2 or TLR4 could have sensed the increased amounts of C. burnetii and caused more cytokine production in the lungs of PMN-depleted mice than in those of the control mice with smaller amounts of bacteria. To further support this idea, the level of a potent chemokine, KC, in the lungs was significantly higher at day 14 p.i. in PMN-depleted mice. The role in clearance from the lungs that neutrophils play could be through direct clearance or through helping other phagocytic cells with clearance.

The next step after bacterial recognition and the production of cytokines and chemokines is the activation and directional migration of neutrophils toward the site of infection. Cytokines of the CXC family, which have a glutamic acid-leucine-arginine (ELR) motif immediately before the CXC sequence, are especially potent chemoattractants for neutrophils (9). CXC cytokines include MIP-1 and KC. When BALF was collected from C. burnetii-infected mice, it was found that PMN-depleted mice had a significantly larger amount of MIP-1 at 7 days p.i. MIP-1 is mainly produced by macrophages when they encounter bacteria and functions to call neutrophils to the site of infection. Since no neutrophils were showing up to the site of infection to help clear the bacteria, the local macrophages likely increased the production of MIP-1 to call more neutrophils. KC levels in the BALF of infected mice were decreased on day 7 p.i. compared to those in the BALF of control mice. After neutrophils travel into the lung and encounter bacteria, they produce KC, which causes more neutrophils to migrate to the site of infection. This explains why the mice depleted of neutrophils would have lower levels of KC.

CXCR2 is a receptor on neutrophils which binds to the highly potent chemokine KC in mice and IL-8 in humans. CXCR2^−/− mice infected intranasally with C. burnetii had similar levels of splenomegaly and similar bacterial burdens in the spleen and lungs. This suggests that this pathway is not necessary for neutrophils to mount an effective immune response to infection. Multiple chemokines which do not act through CXCR2 are produced at the site of infection, including MIP-1, complement factors (C5a), and leukotriene B4 (LTB4). Neutrophils likely used these pathways to migrate into the lung tissue in CXCR2^−/− mice. Mice deficient in CXCR2 still had the same amount of bactericidal enzymes within their granules as mice not deficient in CXCR2, so if they used another pathway to get into the lungs, they were likely able to kill Coxiella, either directly or through helping other phagocytic cells.

Once chemokines bind to their receptors on neutrophils, they normally migrate very rapidly, entering the airway within 24 h following infection with bacteria, such as S. pneumoniae, L. pneumophila, and K. pneumoniae (8–10). However, a previous study from our lab found that following aerosol infection with C. burnetii NMI, neutrophil influx into the lungs is delayed until 7 days postinfection (4). In the current study, spleen size and the bacterial burden in the spleen were similar between PMN-depleted and control mice on day 3 p.i.; however, by day 7, PMN-depleted mice had significantly increased levels of splenomegaly and bacterial burdens compared to those in the control mice. This likely occurs because neutrophils normally do not play a role in C. burnetii infection on day 3. By day 7 p.i., control mice had neutrophil influx into their lungs, but the PMN-depleted mice did not, leading to increased disease severity.

Once neutrophils reach the site of infection, they engulf and kill bacteria. A previous study from our lab found that neutrophils infected with C. burnetii were taken up by macrophages and the bacteria were able to replicate within these macrophages (4). This suggests that neutrophils may not be able to kill C. burnetii directly. It has been shown that C. burnetii uptake by neutrophils decreases the amount of reactive oxygen species (ROS) produced by the neutrophils (24). If the role of neutrophils in C. burnetii infection is not associated with direct killing, there are two other possible roles, based on the increased disease severity observed when neutrophils are depleted: (i) C. burnetii infects neutrophils, which are then taken up by macrophages. C. burnetii bacteria which enter the macrophage through this process are more easily killed than bacteria which are taken up by the macrophage directly. (ii) Activated neutrophils migrate to the lungs, undergo apoptosis, and are taken up by macrophages. The uptake of the highly bactericidal neutrophil granules increases the killing ability of the macrophages. Either of these scenarios would lead to the increased local and systemic disease severity observed in PMN-depleted mice compared to that observed in control mice. In our previous study (4), we did incubate neutrophils overnight in medium and then added them to macrophages for 4 h before infection, and we did not see differences in replication rates. However, activated neutrophils which have traveled from the bloodstream into the lungs are much more effective at killing than neutrophils which are collected from the bone marrow and incubated in medium overnight.

Between days 7 and 14 p.i., the C. burnetii bacterial burden decreased in the lungs and increased in the spleen, suggesting trafficking from the lung to the spleen and clearance from the lungs. However, mice deficient in IL-1R had an increased amount of C. burnetii within the lungs on day 14 p.i. compared to WT mice. The bacterial burden was actually higher on day 14 p.i. than day 7 p.i. This suggests that IL-1 may be involved in the clearance of C. burnetii from the lungs. Recent studies also demonstrated that IL-1 is involved in the clearance of L. pneumophila from the lungs (25, 26). LeibundGut-Landmann et al. found that the IL-1β produced by alveolar macrophages is critical for inducing chemokine production in nonhematopoietic cells, including alveolar epithelial cells. This chemokine production is essential for control of L. pneumophila infection (27). In addition, a study with Legionella pneumophila found that bacterial activation of caspase 11 promotes IL-1β and IL-1α release and increases bacterial clearance (28). Thus, IL-1 may play a critical role in the clearance of bacteria from pulmonary bacterial infections.

It has been shown that IL-17 is an important cytokine for recruiting neutrophils against K. pneumoniae infection in a murine model, and mice lacking the receptor for IL-17 showed an attenuated granulocyte colony-stimulating factor and MIP-1 response, decreased neutrophil recruitment, a greater bacterial burden, and increased mortality (29, 30). To determine whether IL-17 is involved in recruiting neutrophils in response to C. burnetii pulmonary infection, we examined if mice lacking IL-17R would exhibit a difference from WT mice in their ability to defend against C. burnetii intranasal infection. The observation that the level of splenomegaly and the bacterial burden in the spleen and lung were similar between IL-17R^−/− and WT mice suggests that IL-17 may not play an essential role in controlling C. burnetii intranasal infection.

Neutrophils are often perceived to act through a nonspecific innate immune response which is short-lived. However, mounting evidence suggests that neutrophils play an integral role in adaptive immunity. One study found that neutrophils promote the adaptive immune response to Mycobacterium tuberculosis. Neutrophils which have taken up M. tuberculosis are taken up by dendritic cells, and this process helps the dendritic cells initiate naive CD4 T-cell activation (31). The increased level of splenomegaly and bacterial burden found through day 40 p.i. in the current study suggest that neutrophils could play a role in adaptive immune responses following C. burnetii infection. Neutrophils may respond to C. burnetii in a manner similar to that in which they respond to M. tuberculosis, phagocytosing C. burnetii, being taken up by dendritic cells, and then helping with the adaptive immune response. A previous study from our lab found that infected neutrophils are taken up by macrophages (4). It would be very interesting to see if dendritic cells also take up infected neutrophils.

Our results indicate that C. burnetii intranasal infection induced disease that was more severe in PMN-depleted mice than in control mice, suggesting that neutrophils play an important role in host defense against primary C. burnetii infection. However, it is unknown whether neutrophils also play a role in PIV-induced protective immunity. Interestingly, a recent study found in the spleen a specific group of B-cell-helper neutrophils which induced T-cell-independent IgM production by marginal zone B cells (13). To determine whether neutrophils are required for vaccine protection, we examined if depletion of neutrophils in PIV-vaccinated mice during C. burnetii challenge would significantly affect the ability of PIV to confer protection against C. burnetii infection. The observation that PMN-depleted mice had a significantly lower relative body weight and a higher level of splenomegaly than control mice suggests that neutrophils may play an important role in protecting PIV-vaccinated mice from C. burnetii infection-induced severe clinical disease. However, the result that there was no significant difference in bacterial burdens in the lungs and spleen between PIV-vaccinated PMN-depleted and control mice indicates that neutrophils may not play an important role in the clearance of bacteria in PIV-vaccinated mice following infection. Thus, future studies focused on understanding the mechanisms of neutrophil-mediated protection in PIV-induced protection may provide useful information for the development of a new generation of vaccines against Q fever.

The RB6-8C5 antibody has been shown to deplete a subpopulation of inflammatory monocytes, T cells, and dendritic cells (15). These populations could have an impact on the phenotype observed following C. burnetii infection; however, it is unlikely that they are the sole cause of this phenotype. Neutrophils are the first responders to lung infection and could work in concert with these additional cells to fight the infection. Further studies will be needed to find the role of the subpopulation of inflammatory monocytes and T cells.

In conclusion, neutrophils play a role in inflammation and bacterial clearance following intranasal C. burnetii NMI infection. Currently, it is unknown whether this role involves the direct interaction between neutrophils and C. burnetii or if neutrophils act to enhance the immune response of other cells. Furthermore, neutrophils appear to play a role in both early and late immune responses. The exact mechanism by which neutrophils enhance the immune response to C. burnetii infection needs further investigation.