Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and Toll-like receptor 2 knockout mice and protects against reinfection

Abstract

Coxiella burnetii is a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuated C. burnetii phase II (having a truncated "O" chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-gamma(-/-)), BALB/c, Toll-like receptor 2 knockout (TLR2(-/-)), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-gamma(-/-) and TLR2(-/-) mice, which showed elevated rectal temperatures that were sustained for approximately 15 days with transient increases in splenic weights. Reinfection of IFN-gamma(-/-) and TLR2(-/-) mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2(-/-) mouse spleens was observed, greater colonization was evident in the IFN-gamma(-/-) mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-gamma(-/-) macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2(-/-) macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.

FIG. 1.

Infection with C. burnetii NMII provokes greater febrile responses in TLR2^−/− and IFN-γ^−/− mice than in C57BL/6 or BALB/c mice. (A and C) BALB/c and IFN-γ^−/− mice (A) and C57BL/6 and TLR2^−/− (C) mice were infected i.p. with 1 × 10⁸ Coxiella burnetii NMII organisms. Daily measurements of rectal temperatures were performed, showing that infected IFN-γ^−/− and TLR2^−/− mice had statistically higher temperatures than the respective normal strain. *, P < 0.001 for IFN-γ^−/− versus BALB/c and for TLR2^−/− versus C57BL/6 mice. (B and D) Splenic weights of individual mice (five/group/time point) were evaluated at weekly intervals after challenge. At 1 week postinfection, significant increases (*, P < 0.001) in the IFN-γ^−/− and TLR2^−/− splenic weights were observed compared to BALB/c and C57BL/6 mice, respectively. Only BALB/c mice showed a slight increase in their splenic weights at 1 week postinfection. Error bars indicate standard deviations of the means.

FIG. 2.

Reinfection with C. burnetii NMII provokes less severe fever in IFN-γ^−/− mice and no increases in their splenic weights. (A and B) IFN-γ^−/− (A) and BALB/c (B) mice were infected i.p. with 1 × 10⁸ C. burnetii NMII organisms (5/group) or PBS (10/group). Rectal temperatures were measured daily. Thirty days after primary infection, mice were rechallenged with same dose of C. burnetii NMII or PBS, and rectal temperatures were measured. Half of the PBS groups were infected with C. burnetii NMII; the other half were left uninfected. The normal body temperature used as a control to compare febrile responses was between 35.5 and 36.5°C. *, P < 0.001 for Coxiella/Coxiella-reinfected group and for PBS/Coxiella-infected group versus PBS/PBS mice. †, P < 0.001 for PBS/Coxiella- versus Coxiella/Coxiella-reinfected mice. (C) Mice were sacrificed at 10 days after the second challenge and splenic weights measured. Reinfection of IFN-γ^−/− mice provoked increases in rectal temperatures, but these were less severe than after the primary infection. There were no increases in splenic weights of IFN-γ^−/− mice after the second infection. *, P < 0.001 for PBS/Coxiella mice versus PBS/PBS mice and Coxiella/Coxiella-reinfected mice. Data represent the means for individual mice from two experiments ± standard deviations of the means. n.s., not significant.

FIG. 3.

Reinfection with Coxiella burnetii NMII provokes less severe fever in TLR2^−/− mice and no increases in splenic weights. (A and B) TLR2^−/− (A) and C57BL/6 (B) mice were infected i.p. with 1 × 10⁸ C. burnetii NMII organisms (5/group) or PBS (10/group). Rectal temperatures were measured daily. Thirty days after primary infection, mice were rechallenged with same dose of C. burnetii NMII or PBS, and rectal temperatures were measured. Half of the PBS group were infected with C. burnetii NMII, and the other half were left uninfected. The normal body temperature used as a control to compare febrile responses was between 35.5 and 36.5°C. *, P < 0.001 for Coxiella/Coxiella-reinfected versus PBS/PBS mice and for PBS/Coxiella-infected group versus PBS/PBS mice. †, P < 0.001 for PBS/Coxiella- versus Coxiella/Coxiella-reinfected mice. (C) Mice were sacrificed 10 days after the second infection, and splenic weights were measured. As observed with IFN-γ^−/− mice, reinfection of TLR2^−/− mice provoked increases in rectal temperatures, but they were less severe than after the primary infection. No increases in the splenic weights of TLR2^−/− mice were observed after the second challenge. *, P < 0.001 for PBS/Coxiella mice versus PBS/PBS mice and Coxiella/Coxiella-reinfected mice. Data represent the means for individual mice from two experiments ± standard deviations of the means. n.s., not significant.

FIG. 4.

Coxiella burnetii NMII is cleared from spleens of reinfected IFN-γ^−/− and TLR2^−/− mice. Quantitative PCR was performed for C. burnetii detection in spleens from the IFN-γ^−/− and BALB/c mice (A) and TLR2^−/− and C57BL/6 mice (B) depicted in Fig. 2 and 3. C. burnetii NMII in mice was detected by quantitative PCR 10 at days postinfection. The results show that the presence of NMII in spleens of immunodeficient mice infected and reinfected with C. burnetii was reduced significantly (*, P < 0.001 for PBS/Coxiella versus Coxiella/Coxiella groups and for PBS/Coxiella versus PBS/PBS groups). No significant differences were observed in genomic DNA copies in the spleens of once- or twice-infected BALB/c mice; reinfection of C57BL/6 mice did show an increase in genomic DNA copies. †, P < 0.001 for differences between once- or twice-infected BALB/c versus IFN-γ^−/− mice and for once-or twice-infected C57BL/6 versus TLR2^−/− mice. Data represent the means for individual mice from two experiments ± standard deviations of the means.

FIG. 5.

RAW264.7 cells produce proinflammatory cytokines in response to NMII. RAW 264.7 macrophages were infected with C. burnetii NMII at various bacterium-to-macrophage ratios of between 1:1 and 100:1. Macrophages were allowed to adhere overnight, infected, and cultured for 8 h, and supernatants were harvested for cytokine ELISAs. RAW264.7 cells produced increased levels of TNF-α and IL-6 and, to a lesser extent, IFN-γ and GM-CSF. A slight increase in IL-10 was measured, and no production of IL-1α, IL-1β, IL-12p40, IL-12p70, or TGF-β was observed. Samples from 24 h of infection showed a similar trend. Depicted are the means from two experiments ± standard deviations of the means.

FIG. 6.

Peritoneal macrophages from IFN-γ^−/−, but not TLR2^−/−, mice produce proinflammatory IL-6 cytokine. Peritoneal macrophages isolated from C57BL/6, TLR2^−/−, BALB/c, and IFN-γ^−/− mice were infected with C. burnetii NMII at various bacterium-to-macrophage ratios of between 1:1 and 100:1. Data from two individual experiments for each mouse strain infected at a 100:1 ratio are shown. Adherent macrophages were allowed to adhere overnight, infected, and then cultured for 24 h, and supernatants were harvested for cytokine ELISAs. Peritoneal macrophages from C57BL/6 and BALB/c mice infected with NMII produced elevated IFN-γ, TNF-α, GM-CSF, and IL-12p40, with minimal to no IL-6 or IL-12p70. C57BL/6, but not BALB/c, macrophages produced IL-10. IFN-γ^−/− peritoneal macrophages produced significantly increased levels of IL-6, IL-10, IL-12p40, and IL-12p70 compared to BALB/c macrophages. TLR2^−/− macrophages produced only GM-CSF, IL-12p40, IL-12p70, and some IL-10. None of the infected macrophages produced IL-1α, IL-1 β, or TGF-β. Statistical analyses were performed for each described cytokine between infected C57BL/6 versus TLR2^−/− macrophages and between infected BALB/c versus IFN-γ^−/− macrophages. *, P < 0.001. Cytokines were not detected in uninfected macrophages. Error bars indicate standard deviations of the means.

FIG. 7.

Heat-killed Coxiella-specific IgG, IgG1, IgG2a, and IgG2b responses by immunocompetent or immunodeficient mice infected once or twice with C. burnetii NMII. No IgG1 responses were detected in any of the sera of immunocompetent or immunodeficient mice. (A) IgG and IgG2a responses were significantly greater (P < 0.001) in BALB/c mice and IFN-γ^−/− mice infected twice than after a single infection. In IFN-γ^−/− mice, serum levels of IgG2b were significantly greater (P < 0.001) in mice challenged twice with NMII than in mice infected once. IgG and IgG2a antibody responses were significantly greater (P < 0.001) in BALB/c mice than in IFN-γ^−/− mice when mice were infected once or twice with NMII. No significant differences were observed in IgG2b serum levels. (B) IgG, IgG2a, and IgG2b responses were significantly greater (P < 0.001) in C57BL/6 mice infected twice than in those infected once. In TLR2^−/− mice, significant increases in IgG, IgG2a, and IgG2b levels were observed after two infections compared to mice infected once with NMII. IgG levels were significantly reduced compared to those in C57BL/6 mice after a single infection. IgG2a levels in TLR2^−/− mice infected twice with NMII were significantly higher (P < 0.001) than those in C57BL/6 mice infected and subsequently reinfected. By contrast, IgG2b levels in C57BL/6 mice infected twice were significantly greater than those in TLR2^−/− mice infected and reinfected with NMII. Depicted are the means ± standard deviations of the serum IgG end point titers (log₂). *, P < 0.001, for log₂ end point titers of twice- versus once-infected mice. †, P < 0.001 for BALB/c versus IFN-γ^−/− mice and for C57BL/6 versus TLR2^−/− mice.

FIG. 8.

Adoptive transfer of immune lymphocytes prevents fever after infection with NMII in IFN-γ^−/− mice. Immune splenic T and B cells were purified from BALB/c and IFN-γ^−/− mice at 6 weeks after their i.p. infection with C. burnetii NMII or from naive BALB/c and IFN-γ^−/− mice and were adoptively transferred (i.v.) into naive BALB/c or IFN-γ^−/− mice. One day later, mice were challenged with 1 × 10⁸ C. burnetii organisms, and rectal temperatures were measured daily. At 3 and 10 days postchallenge, spleens were harvested and weighed. (A and B) Temperatures (A) and splenic weights (B) on day 3 or 10 postchallenge of naive BALB/c recipients given PBS, lymphocytes from once-infected BALB/c mice, or lymphocytes from naive BALB/c or IFN-γ^−/− mice. (C and D) Temperature (C) and splenic weights (D) on day 3 or 10 postchallenge of naive IFN-γ^−/− recipients given PBS, lymphocytes from once-infected BALB/c or IFN-γ^−/− mice, or lymphocytes from naive IFN-γ^−/− mice. (A and C) *, P < 0.001 for body temperatures of naive IFN-γ^−/− cell- or immunized IFN-γ^−/−cell-treated mice versus PBS-treated mice. (B and D) *, P < 0.001 for splenic weights of PBS-, naive BALB/c cell-, or naive IFN-γ^−/− cell-treated mice versus infected BALB/c or immunized IFN-γ^−/− cell-treated mice at 3 days postchallenge. †, P < 0.001, 10 days postchallenge. Error bars indicate standard deviations of the means.

FIG. 9.

Adoptive transfer of immune lymphocytes prevents fever after infection with NMII in TLR2^−/− mice. Immune splenic T and B cells were purified from C57BL/6 and TLR2^−/− mice 6 weeks after their i.p. infection with C. burnetii NMII or from naive C57BL/6 and TLR2^−/− mice and adoptively transferred (i.v.) into naive C57BL/6 or TLR2^−/− mice. One day later, mice were challenged with 1 × 10⁸ C. burnetii organisms, and rectal temperatures were measured daily. At 3 and 10 days postchallenge, spleens were harvested and weighed. (A and B) Temperatures (A) and splenic weights (B) on day 3 or 10 postchallenge of naive C57BL/6 recipients given PBS, lymphocytes from once-infected C57BL/6 mice, or lymphocytes from naive C57BL/6 or TLR2^−/− mice. (C and D) Temperature (C) and splenic weights (D) on day 3 or 10 postchallenge of naive TLR2^−/− recipients given PBS, lymphocytes from once-infected C57BL/6 or TLR2^−/− mice, or lymphocytes from naive TLR2^−/−. (A and C) *, P < 0.001 for body temperatures of immunized C57BL/6 cell-, naive C57BL/6 cell-, or immunized TLR2^−/−cell-treated mice versus PBS-treated mice. (B and D) *, P < 0.001 for splenic weights of PBS-, naive C57BL/6 cell-, or naive TLR2^−/− cell-treated mice versus immunized C57BL/6 cell- or immunized TLR2^−/− cell-treated mice at 3 days postchallenge. †, P < 0.001, 10 days postchallenge. Error bars indicate standard deviations of the means.

FIG. 10.

Adoptive transfer of immune lymphocytes prevents splenic colonization by Coxiella NMII. Quantitative PCR was performed for C. burnetii detection in spleens of recipient mice used in the adoptive transfer and challenge studies as described for Fig. 8 and 9. Immune or naive splenic T and B cells from BALB/c or IFN-γ^−/− mice were adoptively transferred (i.v.) into naive BALB/c (A) or IFN-γ^−/− (C) recipients; immune or naive T and B cells from C57BL/6 or TLR2^−/− mice were adoptively transferred into naive C57BL/6 (B) or TLR2^−/− (D) recipients. Detection of DNA genomic copies was performed at 3 and 10 days postinfection. (A) BALB/c mice treated with immunized BALB/c or IFN-γ^−/− cells showed a significant reduction in the DNA genomic copies at 10 days postchallenge. (C) Detection in IFN-γ^−/− mice that received immune T and B cells from infected BALB/c or IFN-γ^−/− mice was reduced significantly (P < 0.001) compared to that in PBS- or naive BALB/c cell-treated mice at 3 and 10 days after challenge. (B) No reductions in the Coxiella genomic copies were observed in C57BL/6 recipient mice. (D) In contrast, TLR2^−/− mice that received immune T and B cells from infected C57BL/6 or TLR2^−/− mice showed a significant reduction (P < 0.001) in the presence of Coxiella at 3 and 10 days after challenge. *, P < 0.001 for splenic Coxiella DNA genomic copies in recipient mice given PBS or naive lymphocytes versus recipients given immune lymphocytes at 3 days postchallenge; †, P < 0.001, the same for 10 days postchallenge. Data represent the mean for five mice per time point ± standard deviations of the means.