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Keywords:

  • Coxiella burnetii;
  • infection;
  • aging;
  • granuloma;
  • cytokine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium that lives in myeloid cells (Raoult et al., 2005). After primary infection, only one-half of patients will experience acute Q fever, mainly as a mild, self-limited, flu-like syndrome or more rarely as pneumonia, hepatitis, myocarditis, pericarditidis, meningitis, or encephalitis (Raoult et al., 2005). Coxiella burnetii is also recognized as a potential biological weapon (Madariaga et al., 2003). Epidemiological data clearly show an age-related increase in the incidence and severity of symptomatic infection (Maltezou & Raoult, 2002). Indeed, Q fever has rarely been reported in young children. Although seroprevalence is similar, adolescents are five times more symptomatic following infection than are young children (Maltezou et al., 2004). The relative risk of Q fever is five times higher in the 60–69 year age range than in adults of around 40 years of age (Raoult et al., 2000, 2005). Hence, Q fever is typically a disease of mature adults.

The frequency of certain bacterial infections, including meningitis, tuberculosis and pneumonia, increases strongly with the ageing process (Ruiz et al., 1999; Choi, 2001; Rajagopalan, 2001). The reasons for increased susceptibility to bacterial infections include epidemiological elements, age-associated physiological and anatomical changes, and dysfunction of the immune system (Gavazzi & Krause, 2002). The immune response towards a number of infectious pathogens declines with age, and this decline has been associated with dysregulated functions of macrophages (Ding et al., 1994). With respect to Q fever, the reasons for increased susceptibility of mature adults to bacterial infections remain unclear.

We wondered whether the age-related alterations observed in clinical practice could be reproduced in an experimental model. We hypothesized that ageing is a cornerstone of C. burnetii infection, and compared the infection of young (1 month) and mature (14 months) mice after intraperitoneal injection of C. burnetii organisms. The present study shows that C. burnetii load and granuloma numbers were increased in the spleens of mature mice independently of the microbicidal competence of macrophages.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Université de la Méditerranée. Coxiella burnetii organisms (Nine Mile strain) were cultured as previously described (Leone et al., 2004). Female C57BL/6 mice were purchased from Charles River Labs (L'Arbresle, France) at three weeks of age, kept in a specific pathogen-free mouse facility, and handled according to the rules of Décret No. 87-848 du 19/10/1987, Paris. Sentinel mice did not reveal any infection at the time of experiments. Young and mature mice were inoculated via the peritoneal route with 5 × 105C. burnetii organisms, and were sacrificed at days 4, 7 and 21 postinfection, because the bacterial burden in the liver and spleen of 2–3 month-old mice is detected at day 4 postinfection, is high at day 7, and is undetectable at day 21 (Leone et al., 2004).

Organs were aseptically excised, and tissue samples were embedded in paraffin. All samples were coded, and slides were read in a blinded manner. The presence of bacteria in tissues was revealed by immunohistochemistry, as previously described (Honstettre et al., 2004). Five-micrometer tissue sections were deparaffinized in xylene and rehydrated in graded alcohol. Each tissue section was incubated with anti-C. burnetii rabbit antibodies (at 1/2000 dilution) or normal rabbit serum as a control. Immunodetection was performed with biotinylated anti-rabbit antibodies and peroxidase-labelled streptavidin (Zymed CliniSciences, Montrouge, France) with amino-ethylcarbazole as substrate. After washing, slides were counterstained with Mayer's hematoxylin for 5 min, and bacteria were visualized in tissues as precipitation products. The total number of bacteria detected by light microscopy was determined for each tissue section. The presence of granulomas was studied as previously described (Honstettre et al., 2004). Briefly, 5-μm sections of paraffin-embedded tissues were stained with hematoxylin-eosin-saffron. Granulomas were defined as collections of 10 or more macrophages and lymphocytes. Their number was determined after whole optical examination of at least three tissue sections. They were quantified using the image analyser SAMBA 2005 (SAMBA Technologies, Alcatel TITN, Grenoble, France). Results are expressed as the number of granulomas found per surface unit (mm2).

Peritoneal macrophages were obtained after washing the peritoneal cavity of untreated mice with 10 mL of warm phosphate-buffered saline. Bone-marrow precursor cells were isolated from femurs and cultured in RPMI 1640 containing 25 mM HEPES, 10% fetal bovine serum (Invitrogen, Cergy Pontoise, France) and 15% L-cell-conditioned medium rich in macrophage-colony-stimulating factor, as recently described (Meghari et al., 2007). After culture for 7 days, more than 95% of cells were macrophages, as determined by morphological and phagocytic criteria. Bone marrow-derived macrophages (BMDM) were scrapped and spun down. Peritoneal macrophages and BMDM were plated in 24-well tissue-culture dishes at a density of 2 × 105 well−1.

To determine bacterial replication, macrophages were infected with C. burnetii (bacterium-to-cell ratio of 200 : 1) for 4 h, washed to eliminate free organisms, and cultivated in RPMI 1640 containing 25 mM HEPES and 10% fetal bovine serum for 9 days. Bacterial replication was assessed by quantitative real-time PCR (qPCR), as previously described (Honstettre et al., 2006). Briefly, DNA was extracted using a QIAamp DNA MiniKit (Qiagen), and qPCR was performed using the LightCycler-FastStart DNA Master SYBR Green system (Roche) and carried out with primers FAF216 (5′-GCACTATTTTTAGCCGGAACCTT-3′) and RAF290 (5′-TTGAGGAGAAAAACTGGATGAGA-3′).

Measurements of cytokines and arginase-1 were determined by qualitative real-time reverse transcription-PCR (qRT-PCR), as recently described (Meghari et al., 2007). Coxiella burnetii organisms (50 : 1 bacterium-to-cell ratio) were added to BMDM for 4 h, and total RNA was purified with Trizol (Invitrogen). Genomic DNA was eliminated by treatment with RNase-free DNase (Qiagen). RNA quality was assessed using a 2100 Bioanalyzer and RNA 6000 Nano LabChip kit (Agilent Technologies), and RNA was stored at −20°C until use. Reverse transcription (Superscript II, Invitrogen) of 10 ng of RNA was performed with oligo(dT) and specific primers for cytokines, arginase-1, and β-actin, all of which were designed using the primer3 tool available at the website http://frodo.wi.mit.edu/cgi-bin/primer3/primer_www.cgi. Reverse transcriptase was omitted in negative controls. The fold change in target gene cDNA relative to the β-actin endogenous control was determined with the formula: fold change=2−ΔΔCt, where ΔΔCt=(CtTarget−CtActin)stimulated−(CtTarget−CtActin)unstimulated.Ct values were defined as the cycle numbers at which fluorescence signals were detected. We considered gene expression to be modulated if it exhibited a fold change>2.0.

Results are expressed as median or mean±SD, and compared using the Mann–Whitney U test. Differences were considered significant when P<0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Young and mature C57BL/6 mice were inoculated with 5 × 105C. burnetii organisms via the peritoneal route: mortality and morbidity were not observed up to 28 days. In addition, there was no difference in physical appearance between young and mature mice, suggesting that age did not modify the resistance of mice towards C. burnetii. Bacteria were detected as red rods in granulomatous infiltrates in the spleen of young and mature mice at day 7 postinfection, and became undetectable at day 21. At day 7, the number of bacteria found in the spleen was significantly higher in mature than in young mice (4.45 vs. 1.04 organisms per mm2, P<0.0001) (Fig. 1a). Bacteria were also detected, but to a low extent, in the liver of mature and young mice at day 7 postinfection (0.08±0.02 and 0.06±0.03 organisms mm−2, respectively). Coxiella burnetii organisms were not found in the lungs, heart or mesenteric lymph nodes of young and mature mice up to 21 days.

image

Figure 1. Coxiella burnetii burden in mice. Young and mature mice were infected with C. burnetii for 7 days. (a) Bacteria in the spleen were revealed by immunostaining. The figure shows the median number of bacteria; data are the average of 10 mice. (b–d) Splenic granulomas were revealed by histochemical analysis and quantified by image analysis. (b) Median no. of granulomas; data are the average of 10 mice. (c) and (d) Show representative micrographs of granulomas in young and mature mice, respectively.

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As C. burnetii organisms were detected within granulomas, we numbered splenic granulomas in young and mature mice. In mature mice, granulomas were detected after 4 days; their number increased at day 7 postinfection and decreased at day 21 postinfection. In young mice, the number of granulomas was low whatever the time course of C. burnetii infection. It was significantly lower (0.92±0.22 vs. 3.35±0.99 granulomas mm−2, P<0.0001) than in mature mice at day 7 postinfection (Fig. 1b). Granulomatous reactions were focal and scattered throughout splenic red pulp. The appearance and composition of granulomas were similar in young (Fig. 1c) and in mature (Fig. 1d) mice. They were mainly composed of macrophages and lymphocytes, with few polymorphonuclear leukocytes.

As the formation of granulomas is controlled by cytokines, we measured the expression of mRNA encoding cytokines and arginase-1 after 4 h of C. burnetii stimulation of BMDM. BMDM from young mice showed marked mRNA expression of inflammatory cytokines [tumor necrosis factor (TNF), IP-10, interleukin (IL)-18, IL-23 and IL-12p40], with a fold change higher than 2.0 (Fig. 2a). They also overexpressed markers associated with an anti-inflammatory profile [IL-10, transforming growth factor (TGF)-β, arginase-1] (Fig. 2b). In contrast, C. burnetii-stimulated expression of mRNA for inflammatory cytokines (Fig. 2a) and anti-inflammatory (Fig. 2b) markers was down-modulated in BMDM from mature mice.

image

Figure 2. Coxiella burnetii infection of macrophages. BMDM were infected or not infected with C. burnetii for 4 h. Total RNA was extracted and transcripts of cytokines (a) and arginase-1 (b) were quantified by qRT-PCR. Results are expressed as the ratio of expression levels in infected BMDM vs. uninfected BMDM. They represent the mean±SD of four experiments. Peritoneal macrophages (c) and BMDM (d) from young and mature mice were infected with C. burnetii, and the infection was followed for 9 days. Bacterial DNA copy number was determined by qPCR. Data are the average of three mice per time point.

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Finally, peritoneal macrophages and BMDM were infected with C. burnetii at a 200 : 1 bacterium-to-cell ratio, and infection was evaluated at days 0, 3, 6, and 9 by quantifying C. burnetii DNA. The number of bacterial DNA copies decreased similarly in peritoneal macrophages from mature and young mice (Fig. 2c), showing that the microbicidal competence of peritoneal macrophages was similar in mature and young mice. BMDM were unable to kill C. burnetii because the number of C. burnetii DNA copies remained constant, but, again, there was no difference between mature and young mice (Fig. 2d).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Our results clearly show that C. burnetii infection was age-dependent, because bacterial burden in the spleen was higher in mature than in young mice. Our results are consistent with those obtained with Listeria monocytogenes (Patel, 1981). The pattern is slightly different with Mycobacterium tuberculosis. Indeed, in the spleens of 3-month-old mice infected with M. tuberculosis, a period of progressive bacterial growth and progressive elimination is followed by a phase of chronic or apparently slowly recrudescing disease. This profile is also observed in 12-month-old mice, whereas the second phase is no longer evident in 24-month-old mice (Orme, 1987). In fact, this divergence reflects epidemiological data. Tuberculosis is a disease of the elderly (Rajagopalan, 2001), whereas Q fever is a disease of mature adults (Raoult et al., 2000). Hence, ageing should be considered in murine models in order to reproduce the epidemiology of infectious diseases.

As increased C. burnetii burden suggests that the response to C. burnetii infection is probably impaired in mature mice, we studied granuloma expression in young and mature mice. Liver granulomas (data not shown) and splenic granulomas were mainly composed of macrophages, lymphocytes and a few polymorphonuclear cells, and were detected in liver lobules, portobiliary spaces (data not shown) and splenic red pulp. Granuloma formation, which reflects the development of cell-mediated immunity (Sandor et al., 2003), was increased in the spleen of mature as compared to young mice. Interestingly, evidence shows that natural killer cells, the cytotoxicity of which decreases during aging (Mocchegiani & Malavolta, 2004), play a direct regulatory role in granulomatous inflammation (Hashimoto et al., 1990).

As interferon-γ, TNF, and IL-12/IL18 have been demonstrated to be involved in the formation of granulomas (Kaufmann, 1999), we investigated the production of cytokines after C. burnetii stimulation of BMDM. Results suggest that the formation of granulomas in mature mice is not related to an anti-inflammatory ability of macrophages to respond to C. burnetii challenge with, as a consequence, bacterial replication. They suggest, rather, that macrophages from young mice were fully reactive to C. burnetii, whereas macrophages from mature mice were refractory to C. burnetii challenge.

Finally, we wondered if increased C. burnetii burden in mature mice was the result of impaired microbicidal competence of macrophages, because C. burnetii lives in macrophages (Raoult et al., 2005). No differences were observed in terms of microbicidal competence between macrophages from mature and young mice. These results are in agreement with a model of M. tuberculosis infection, demonstrating that macrophages were fully functional in aged mice (Rhoades & Orme, 1998). Hence, the ability of macrophages to clear bacteria appears to be independent of age. Together with the role of sex hormones previously reported in C. burnetii infection (Leone et al., 2004), these results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.

In conclusion, we developed an animal model in which age is associated with increased tissue infection, granuloma formation and defective responses to bacterial stimulation. This model is useful for understanding why mature adults are at risk of developing symptomatic Q fever.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Programme Hospitalier de Recherche Clinique 2005, ‘Immunosuppression de la fièvre Q: rôle des cellules régulatrices et de l'apoptose’.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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