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. 2015 Apr;81(4):234-9.
doi: 10.1016/j.diagmicrobio.2014.12.008. Epub 2014 Dec 30.

Early cytokine and antibody responses against Coxiella burnetii in aerosol infection of BALB/c mice

Teske Schoffelen  1 , Joshua S Self  2 , Kelly A Fitzpatrick  2 , Mihai G Netea  1 , Marcel van Deuren  1 , Leo A B Joosten  1 , Gilbert J Kersh  3
Affiliations

Early cytokine and antibody responses against Coxiella burnetii in aerosol infection of BALB/c mice

Teske Schoffelen et al. Diagn Microbiol Infect Dis. 2015 Apr.

Abstract

Coxiella burnetii, a Gram-negative intracellular bacterium, can give rise to Q fever in humans and is transmitted mainly by inhalation of infected aerosols from animal reservoirs. Serology is commonly used to diagnose Q fever, but the early cellular immune response-i.e., C. burnetii-specific interferon γ (IFN-γ) production in response to antigen challenge-might be an additional diagnostic. Detection of IFN-γ responses has been used to identify past and chronic Q fever infections, but the IFN-γ response in acute Q fever has not been described. By challenging immunocompetent BALB/c mice with aerosols containing phase I C. burnetii, the timing and extent of IFN-γ recall responses were evaluated in an acute C. burnetii infection. Other cytokines were also measured in an effort to identify other potential diagnostic markers. The data show that after initial expansion of bacteria first in lungs and then in other tissues, the infection was cleared from day 10 onwards as reflected by the decreasing number of bacteria. The antigen-induced IFN-γ production by splenocytes coincided with emergence of IgM phase II antibodies at day 10 postinfection and preceded appearance of IgG antibodies. This was accompanied by the production of proinflammatory cytokines including interleukin (IL) 6, keratinocyte-derived cytokine, and IFN-γ-induced protein 10, followed by monocyte chemotactic protein 1, but not by IL-1β and tumor necrosis factor α, and only very low production of the anti-inflammatory cytokine IL-10. These data suggest that analysis of antigen-specific IFN-γ responses could be a useful tool for diagnosis of acute Q fever. Moreover, the current model of C. burnetii infection could be used to give new insights into immunological factors that predispose to development of persistent infection.

Keywords: Cellular immunity; Coxiella burnetii; Cytokines; Interferon-gamma; Q fever; Serology.

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Conflict of interest statement

Conflict of interest

A patent application has been submitted by MGN, MVD and LABJ for a C. burnetii–specific IFN-γ production assay to diagnose Q fever and is registered by the number PCT/NL 2011/050564.

Figures

Figure 1
Figure 1. Spleen weight after aerosol infection with C. burnetii in immunocompetent BALB/c mice
(A) Spleen-to-body weight (mean ± SD) is shown for 10 uninfected mice (t=0) and 8 infected mice per time point post-infection. ANOVA test followed by Dunn’s multiple comparison test was used to compare infected mice at different time points with uninfected mice. **, P<0.01; ***, P<0.001.
Figure 2
Figure 2. Number of C. burnetii DNA copies in lung, plasma, liver and spleen after aerosol infection with C. burnetii in immunocompetent BALB/c mice
The mean ± SD numbers of genomic equivalents per gram of tissue or ml of plasma are shown of 8 infected mice per time point. Uninfected mice were negative at every time point in all tissues. Samples that were negative were assigned a value of 200 genomic equivalents per g (or ml). This is the limit of detection of the assay. P values were calculated by one-sample t-test with a hypothetical value of 0. * P<0.05, ** P<0.01, *** P<0.001.
Figure 3
Figure 3. Antibody responses to C. burnetii after aerosol infection with C. burnetii in immunocompetent BALB/c mice
IgM and IgG titers to Nine Mile phase I and phase II were measured in plasma by indirect immunofluorescence assay (IFA). The median ± range reciprocal titers are shown of four pairs of infected mice per time point. The control mice were seronegative at every time point (not shown). Negative results in the IFA were assigned a value 1:8.
Figure 4
Figure 4. Early IFN-γ production by stimulated splenocytes of C. burnetii-infected immunocompetent BALB/c mice
Splenocytes were stimulated for 48h with either conA [2.5 µg/mL], NM phase I [10^7/mL], NM phase I [10^6/mL], or left unstimulated (nil). The median ± IQR cytokine production is shown per time point of four pairs of infected mice. T=0 shows the median ± IQR of five pairs of uninfected mice. P values were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparison test comparing cytokine concentrations of infected mice at different time points with uninfected mice. * P<0.05, ** P<0.01, *** P<0.001. Abbreviations: conA, concanavalin A; NMI, C. burnetii Nine Mile phase I.
Figure 5
Figure 5. Early IL-6, KC, MCP-1, IP-10 and IL-10 production by stimulated splenocytes of C. burnetii-infected immunocompetent BALB/c mice
Splenocytes were stimulated for 48h with either conA [2.5 µg/mL], NM phase I [10^7/mL] or [10^6/mL], or left unstimulated (nil). The median ± IQR cytokine production is shown per time point of four pairs of infected mice. T=0 shows the median ± IQR of five pairs of uninfected mice. IL-1β and TNF-α production were below the lowest detection limit at all time points for all stimulations (not shown).The dashed horizontal line represents the lowest standard in the Luminex assay, values below are extrapolated. P values were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparison test comparing cytokine concentrations of infected mice at different time points with uninfected mice. * P<0.05, ** P<0.01. Abbreviations: conA, concanavalin A; NMI, C. burnetii Nine Mile phase I.
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