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. 2001 Mar;123(3):428–434. doi: 10.1046/j.1365-2249.2001.01474.x

Effect of oestrogen on Mycobacterium avium complex pulmonary infection in mice

K Tsuyuguchi 1, K Suzuki 1, H Matsumoto 1, E Tanaka 1, R Amitani 1, F Kuze 1
PMCID: PMC1906003  PMID: 11298130

Abstract

The purpose of the present study was to elucidate the role of oestrogen in the pathogenesis of Mycobacterium avium complex (MAC) pulmonary disease, which occurs most frequently in postmenopausal women. The study was carried out in a murine infectious model using ovariectomized DBA/2 female mice. Infection with MAC was established by intratracheal administration of bacilli. In some experiments, ovariectomized mice were treated with exogenous 17β-estradiol (E2). The number of bacilli in the lungs of infected mice which received ovariectomy was significantly larger than that in the lungs of sham-operated control mice, and treatment of ovariectomized mice with exogenous E2 restored the burden of bacilli to the same level as that in the sham-operated control mice. We next examined the effect of E2 in vitro using bone marrow-derived macrophages obtained from DBA/2 female mice. The macrophages showed bacteriostatic activity against MAC after treatment with interferon-gamma (IFN-γ) and this activity was further enhanced by the exogenous addition of E2 to the culture medium. In parallel with these findings, E2 augmented the production of reactive nitrogen intermediates (RNI) by macrophages pretreated with IFN-γ and stimulated with MAC, as shown by evaluating nitrite production and inducible nitric oxide synthase mRNA expression. These findings taken together suggest that absence of endogenous oestrogen appears to be responsible for the development of MAC pulmonary disease in this mouse model and that the enhancement by E2 of anti-MAC activity of murine macrophages induced through increased RNI production may play some role in resistance to MAC infection.

Keywords: Mycobacterium avium complex (MAC), oestrogen, pulmonary infection

INTRODUCTION

The incidence of Mycobacterium avium complex (MAC) disease is steadily increasing globally [1–3]. In addition to disseminated cases, which often occur in AIDS patients, pulmonary disease in patients without obvious predisposing conditions is becoming an important clinical problem. This type of disease is characterized by nodules and/or bronchiectasis on CT scans, and is also characterized by its preponderance in postmenopausal women [2–4]. As much as 80% to 90% of the cases occur in women, most of whom are elderly. Menopause normally occurs between the ages of 42 and 60 years, accompanied by the decline in the production of oestrogen and progesterone [5]. These suggests the involvement of sex hormone fluctuations in the establishment of this type of pulmonary MAC disease.

Oestrogen, a major female sex hormone, has been reported to modulate resistance to a variety of infections. Oestrogen diminishes the severity of infections by some pathogens [6], whereas it enhances susceptibility to other pathogens [7,8]. These divergent effects of oestrogen may be partly due to its divergent effects on various immune functions such as macrophage activity [6,9,10], natural killer cell activity [11] and T-cell mediated immunity [12,13].

The purpose of the present study was to evaluate how endogenous oestrogen modulates pulmonary MAC infection in an experimental murine model mimicking this type of human pulmonary MAC disease. We observed that ovariectomy of DBA/2 female mice with intratracheal MAC infection resulted in a larger burden of the organisms in the lungs compared with that in control animals. This higher burden of infection in the ovariectomized animals was abrogated by the administration of exogenous oestrogen. We also found that oestrogen enhanced the anti-MAC activity of murine macrophages by modifying reactive nitrogen intermediates (RNI) production, which may partly explain the effects of oestrogen in vivo.

MATERIALS AND METHODS

Mice

All mice used for this study were specific-pathogen-free DBA/2 female mice obtained from Japan SLC Inc., Shizuoka, Japan. Animals were 6-week-old and weighed an average of approximately 20 g at the time of these studies.

Bacteria

M. avium Mino (kindly provided by Dr R. M. Nakamura, Division of Mycobacteriology, National Institute of Health, Tokyo), originally isolated from the sputum of a patient with pulmonary MAC disease, was grown in modified Dubos Tween-albumin liquid medium (Difco Laboratories, Detroit, USA) and 1-ml aliquots of the bacterial suspension were stored in separate tubes at − 80°C until use. Before use, the frozen sample was quickly thawed, washed once and sonicated for 2 min using a microtip sonicator (Handy Sonic UR-20, TOMY SEIKOSHA, Tokyo, Japan) to disperse large bacterial clumps. The bacterial suspension thus obtained was diluted with physiological saline and adjusted to the desired titre.

Experimental infection

Infection of mice with MAC was conducted intratracheally. Mice were anaesthetized with pentobarbital and the trachea was visualized by a ventral midline cervical skin incision. Fifty microliters of bacterial suspension containing 2 × 105 CFU of MAC was inoculated into the trachea using a 26-gauge needle. At regular intervals after infection, mice were sacrificed and the numbers of viable bacilli in lungs and spleens were estimated. These organs were aseptically removed, weighed and homogenized with a tissue homogenizer (Universal Homogenizer; Nippon Seiki, Tokyo, Japan). Serial 10-fold dilutions of the organ homogenates were inoculated onto Middlebrook 7H10 agar plates and incubated at 37°C in 5% CO2. After incubation for 10 days, the numbers of CFU were counted to determine the number of viable bacilli per organ.

Ovariectomy and oestrogen treatment

Ovaries of 6-week-old female mice were removed through bilateral incisions over the dorsum under pentobarbital anaesthesia. The incisions were closed by suture. For sham-operation, the ovaries were identified and the wound then closed. All mice were infected with MAC 3 weeks after the operation. For the oestrogen restoration experiment, mice were treated with 17β-estradiol (E2) (Sigma Chemical Co.,St. Louis, MO.). The chemical was dissolved in corn oil by gentle heating. Mice were injected subcutaneously with E2 (0·1 mg/kg) or vehicle (corn oil only). Treatment was done three times a week beginning one week before infection with MAC and ending just before sacrifice.

In vitro macrophage cultures

To obtain bone marrow-derived macrophages (BMMφ), femurs and tibias from 6- to 8-week-old DBA/2 female mice were removed and the marrow was flushed with 10 ml of Hank's balanced salt solution using a 25-gauge needle. The cell suspension was washed and cultured at 37°C in 5% CO2 in phenol red-free RPMI 1640 (Gibco-BRL, Gaithersburg, Md.) supplemented with 10% (v/v) foetal bovine serum (FBS) (Gibco-BRL), 104 U/ml of M-CSF (kindly provided by Yoshitomi Pharmaceutical Co., Osaka, Japan) and HEPES buffer. No antibiotics were used. At 5 days after seeding, the attached cells were rinsed with phosphate buffered saline (PBS) and re-fed with fresh medium containing 104 U/ml of M-CSF and various concentrations of E2 (0, 10, 100 ng/ml).

Infection of macrophage monolayers with MAC

On day 10 after seeding, macrophage cultures were stimulated with murine recombinant interferon-gamma (rIFN-γ; Genzyme Co., Cambridge, MA) or not stimulated. After 24 h, cells were infected with MAC at a mycobacterium/macrophage ratio of 1 : 1 for the kinetic studies of bacterial replication or 10 : 1 for the nitrite assay or competitive RT-PCR analysis.

Quantification of intracellular growth of MAC

After infection for 6 h, macrophage layers were washed using warm PBS to eliminate noningested bacteria, and re-fed with fresh medium. At this time and 4 days after infection, the numbers of viable intracellular bacilli were determined after lysis of the macrophages and culturing on 7H10 agar plates. To lyse macrophages, sterile water was added to each well, and the mixtures were incubated for 10 min and sonicated for 10 s using a microtip sonicator.

Nitrite assay

To assess the amount of nitric oxide (NO) produced, the culture supernatants were assayed to determine the concentration of nitrite, a stable end product of NO. Nitrite was measured using the Griess reaction, as previously described [14]. Briefly, supernatants (50 μl) were incubated with equal volumes of the Griess reagent (1% sulfanilamide, 2·5% H3PO4, 0·1% N-(1-naphthyl)ethylenediamine dihydrochloride) and incubated for 10 min at room temperature, and the optical density was read at 540 nm. The concentration of nitrite was determined using sodium nitrite as a standard.

RNA isolation, cDNA synthesis and RT-PCR analysis

Total RNA from macrophages or organs was extracted using the guanidinium-isothiocyanate method with Trizol (Gibco-BRL). From the RNA, cDNA was synthesized using the standard reverse transcription (RT) method [15]. Aliquots of cDNA were amplified using PCR with oligonucleotide primers specific for β-actin, IFN-γ and iNOS. The sequences of the primers for β-actin (349 bp), IFN-γ (242 bp) and iNOS (479 bp) have been published previously [16]. A GeneAmp PCR system 9600-R (Perkin-Elmer Cetus Inc., Norwalk, Conn.) was used to run 30 cycles of denaturation at 92°C for 40 s, annealing at 60°C for 40 s, and extension at 75°C for 90 s. PCR products were electrophoresed on 1·6% agarose gels (Sigma) and stained with ethidium bromide.

Competitive RT-PCR analysis

To compare separate samples semiquantitatively, competitive PCR was performed. One set of gene-specific primers was used to amplify both the target cDNA and control DNA fragment (the internal standard). Control DNAs were constructed using a PCR MIMIC construction kit (Clontech Laboratories Inc., Palo Alto, Calif.) with the gene-specific primers described above. The size of the PCR product of the control DNA was 606 bp. Each target DNA was amplified in the presence of serial 10-fold dilutions of control DNA fragments to estimate the amount required to achieve equal band intensities for both fragments. To determine the level of mRNA expression in the organs, aliquots of cDNA were diluted 10-fold and control fragments were added at dilutions ranging from 1 to 10−2 amol/ml for β-actin and from 10−1 to 10−3 amol/ml for IFN-γ. To determine the level of mRNA expression in the macrophages, aliquots of cDNA were diluted 10-fold and the control fragments were added at dilutions ranging from 10 to 10−1 amol/ml for β-actin and from 10-2 to 10-4 amol/ml for iNOS. Competitive PCR was performed as described in the manufacturer's instructions. PCR products were electrophoresed on 1·6% agarose gels (Sigma) and stained with ethidium bromide. It was then determined which 10-fold serial dilution gave target and control DNA bands of equal intensities. The determined concentration (in amol/μl) of the control DNA was regarded as the amount of the target DNA. If there were no bands with equal intensity, the amount of the target DNA was considered to lie between the two serial 10-fold-diluted concentrations of control DNA for which there was the least difference between the target and control DNA bands.

Statistical analysis

Significance levels were determined by a one-way analysis of variance and means were compared using Fisher's PLSD test; a P-value of less than 0·05 was considered to be significant.

RESULTS

Effect of E2 on the growth of MAC in vivo

To assess whether administration of E2 could influence MAC growth in vivo, we first used intact (without ovariectomy) DBA/2 female mice and evaluated the effect of E2. The lung weight and the bacterial CFU in the lung did not differ significantly between the E2-treated group and control group (data not shown). We considered it likely that the lack of effect of exogenous oestrogen might have been due to endogenous oestrogen in the mice. Therefore, we next evaluated the effect of E2 in ovariectomized mice. Mice were subjected to ovariectomy or sham-operation 3 weeks before infection with MAC. Ovariectomized mice received treatment of E2 (OVX + E) or vehicle (OVX + C) and sham-operated mice received that of vehicle (Sham + C). As shown in Fig. 1a, a two-fold increase in lung MAC count was observed in ovariectomized mice compared to that found in sham-operated control mice assessed on either day 21 or day 42 after infection. Treatment of ovariectomized mice with E2 reduced the burden of bacilli in the lungs to the same level as that in sham-operated control mice. As shown in Fig. 1b, lungs of vehicle-treated ovariectomized mice weighed more than those of E2-treated ovariectomized or sham-operated control mice, which is consistent with the results of CFU data. These results suggest that an E2-deficient state in ovariectomized mice might be responsible for the increased number of MAC in the lungs. No bacterial growth was observed in the spleens of mice on day 1, and the CFU counts in the spleens on either day 21 (OVX + E 6·0 × 102 ± 3·5 × 102; OVX + C 1·9 × 103 ± 1·1 × 103; Sham + C 5·0 × 102 ± 2·2 × 102) or day 42 (OVX + E 1·2 × 104 ± 4·7 × 103; OVX + C 1·6 × 104 ± 8·1 × 103; Sham + C 6·8 × 103 ± 3·9 × 103) remained considerably lower than those in the lungs and there were no significant differences among the groups. This might be explained by the use of the intratracheal rather than the intravenous route of administration of bacilli in the present study.

Fig. 1.

Fig. 1

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Changes in (a) the CFU in the lungs and (b) the lung weights of vehicle-treated ovariectomized mice (OVX + C, □), E2-treated ovariectomized mice (OVX + E2, ○) and vehicle-treated sham-operated mice (Sham + C, •). Mice were operated on 3 weeks before infection and then infected intratracheally with MAC Mino strain. Each value is a mean ± SEM from groups of eight mice. *P < 0·05 compared with OVX + C; **P < 0·01 compared with OVX + C; ***P < 0·005 compared with OVX + C.

Effect of E2 on the growth of MAC in BMMφ

We next determined the direct effects of E2 on MAC growth by seeding MAC into RPMI 1640 containing FBS with various concentrations of E2 (0, 10, 100 ng/ml) and evaluating the CFU counts on either day 7 or day 14 after seeding. The presence of E2 did not affect the MAC growth in cell free medium (data not shown), which suggested that E2 exerted its effects on the MAC growth in vivo by modifying host resistance. It is generally accepted that host resistance against mycobacteria depends largely on the killing by macrophages activated by various cytokines, especially IFN-γ, which is mainly produced by T cells and natural killer cells [17–19]. Thus, we first evaluated the effect of oestrogen on the production of IFN-γ by comparing the level of IFN-γ mRNA expression in the lungs and spleens of E2-treated mice and untreated mice by competitive RT-PCR analysis. We found the same strong expression of IFN-γ mRNA in the two groups of mice, regardless of oestrogen status (data not shown). We therefore focused our attention on macrophage function as a target of E2 and performed a series of experiments using murine BMMφ. First, we evaluated the effect of E2 on the growth of MAC in macrophages. As shown in Fig. 2, pretreatment with IFN-γ induced significant bacteriostasis of BMMφ against MAC, and the addition of exogenous E2 to the culture media induced maximally two-fold reduction in bacterial count, which was significantly greater than IFN-γ alone. The presence of E2 alone, however, did not affect the intracellular growth of MAC. There was no significant difference in lactate dehydrogenase concentration in the culture supernatants of BMMφ subjected to the various treatments (control 141·3 ± 5·2; E2(10 ng/ml) 136·3 ± 4·5; E2(100 ng/ml) 138·7 ± 2·6; control + IFN-γ 131·7 ± 3·5; E2(10 ng/ml) + IFN-γ 131·7 ± 3·5; E2(100 ng/ml) + IFN-γ 137·3 ± 4·8 U/ml, P > 0·1), which suggested that the difference in CFU counts was not due to a difference in the cell viability. Thus, our findings indicated an enhancing effect of E2 on the antimirobial activity of IFN-γ-activated murine BMMφ in vitro.

Fig. 2.

Fig. 2

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Effect of E2 on the growth of MAC in cultured murine bone marrow macrophages. Macrophages cultured with various concentrations of E2 were pretreated for 24 h with IFN-γ (100 U/ml) or not and then each cell was infected with MAC. CFU counts at 2 days after infection are shown. Each value is a mean ± SEM from groups of five wells. *P < 0·0005 compared with control; **P < 0·005 compared with IFN-γ-pretreated cells without E2.

E2 increases NO production by BMMφ

RNI have been shown to be involved in the killing of mycobacteria by infected macrophages [20–24]. To determine whether E2 affects NO production by macrophages, BMMφ cultured in the presence of various concentrations of E2 were pretreated with IFN-γ and then infected with MAC. On day 4 after MAC infection, supernatants of the cells were assayed to determine the concentration of nitrite. As shown in Fig. 3, the presence of E2 in the culture medium caused significant enhancement of nitrite production by IFN-γ-pretreated, MAC-stimulated BMMφ in a dose-dependent manner. E2, however, did not induce nitrite production by BMMφ in the absence of IFN-γ and MAC (data not shown).

Fig. 3.

Fig. 3

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Effect of E2 on the nitric oxide production of cultured murine bone marrow macrophages. Macrophages cultured with various concentrations of E2 were pretreated for 24 h with IFN-γ (100 U/ml) and then each cell culture was infected with MAC. Cells not treated with E2 or IFN-γ were also infected with MAC. Supernatant nitrite concentrations at 4 days after infection are shown. Each value is the mean ± SEM from four wells.*P < 0·0001 compared to cells not treated with E2 or IFN-γ; **P < 0·05 compared to IFN-γ-pretreated cells not treated with E2.

E2 augments iNOS mRNA expression in BMMφ

We next evaluated the effects of E2 on the expression of iNOS mRNA in BMMφ. To determine when the peak level of iNOS mRNA expression occurred, BMMφ pretreated with IFN-γ were stimulated with MAC for 2, 6, 12, or 24 h, and RT-PCR analysis was performed. Because peak levels of the message were achieved at 12 h after stimulation with MAC (data not shown), subsequent experiments were performed at this time point.

BMMφ cultured in the presence of various concentrations of E2 were pretreated with IFN-γ and stimulated with MAC as described earlier, and total cellular RNA was isolated 12 h after stimulation with MAC for competitive RT-PCR analysis. As shown in Fig. 4, PCR samples consistently contained about 1 amol of β-actin per ml. Pretreatment with IFN-γ and stimulation with MAC induced the expression of iNOS mRNA, while macrophages without the pretreatment and the stimulation, and macrophages stimulated with MAC alone did not express iNOS mRNA. Moreover, as shown in lane 5 of Fig. 4, the addition of E2 resulted in about a 10-fold to 102-fold increase in IFN-γ-and MAC-induced iNOS mRNA expression. These findings further indicated that E2 augments RNI-mediated antimicrobial activity of macrophages against MAC, which may partly account for the difference of bacterial burden in the lung depending on the oestrogen status in vivo.

Fig. 4.

Fig. 4

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Results of competitive RT-PCR analysis of iNOS mRNA expression in macrophages. Aliquots of cDNA were coamplified with 10-fold serial dilutions of control fragments. Lanes: 1, unstimulated macrophages; 2, macrophages stimulated with MAC without IFN-γ pretreatment; 3, macrophages pretreated with IFN-γ and stimulated with MAC; 4, macrophages cultured with 100 ng/ml of E2 and stimulated with MAC without IFN-γ pretreatment; 5, macrophages cultured with 100 ng/ml of E2 and pretreated with IFN-γ and stimulated with MAC. The data shown are representative of three independent experiments with similar results.

Discussion

The typical patient with pulmonary MAC disease had some underlying lung disease. Hence, it was suggested that healthy humans seldom suffered from pulmonary MAC disease and that some defect in the host defence mechanisms lead to the establishment of pulmonary MAC disease. Recent reports, however, have noted a preponderance of elderly women MAC patients without obvious predisposing conditions [2–4]. We hypothesized that female sex hormones might play some role in protection against pulmonary MAC infection, since the serum concentrations of female sex hormones in women are reduced postmenopausally while those in men are constant throughout life.

The purpose of the present study was to evaluate how oestrogen, one of the female sex hormones, is involved in the establishment of pulmonary MAC disease using an experimental murine system. In the present study, we established a murine infection model to mimic human pulmonary MAC disease: DBA/2 strain mice, which was shown to be resistant to intravenous [25] or intraperitoneal [26] MAC infection, were employed throughout the experiments. Infection was carried out by intratracheal administration of MAC. Experimental menopause was produced by ovariectomy of 6-week-old female mice. We found that using this model, MAC replicated mainly in the lungs and few MAC grew in the spleens, which is consistent with human pulmonary MAC disease. The findings of the present study demonstrated that in ovariectomized mice larger numbers of MAC were found in the lungs than in control mice, and that exogenously administered oestrogen restored the level of MAC in the lungs of the ovariectomized mice to that in the sham-operated mice.

There have been some reports about the relation between sex and mycobacterial infections using experimental murine infection, such as those due to M. intracellulare [27], M. marinum [28] and M. leprae [29]. They all showed that females were more resistant to the infections than males. It is possible that oestrogen exerted positive protection effects, but none of the reports examined that possibility. To our knowledge, the present study is the first to demonstrate the relation of oestrogen to the severity of pulmonary MAC infection using an ovariectomized murine model.

The role of oestrogen in host resistance against various microbial infections has been extensively examined. There are, however, discrepancies in the conclusions drawn from these studies about the effect of oestrogen on host defense. Oestrogen was reported to increase resistance to some pathogens such as Salmonella, Streptococcus and Pasteurella species [6]. Conversely, oestrogen could make some animals more vulnerable to listerial and gonococcal infections [7,8]. Oestrogen affects various immune functions including macrophage activity [6,9,10], natural killer cell activity [11] and T-cell mediated immunity [12,13] and oestrogen often exhibits heterogenous effects on functions such as tumour necrosis factor-α production by macrophages [9]. This diversity in the effects of oestrogen on immune functions may account for the discrepant findings about the susceptibility against some microbial pathogens described above.

Our series of experiments suggest that oestrogen augments the microbicidal activities of murine macrophages against MAC through, at least in part, enhancement of RNI production. There is abundant evidence showing the importance of RNI as a major mediator of the microbicidal activity of murine macrophages against mycobacteria [20,21] and its role in human macrophages has also been shown in recent reports [22–24]. Neugarten et al. showed that oestrogen increased renal medullary iNOS levels in ovariectomized female rats, which is consistent with the results of the present study [30], whereas Hayashi et al. showed that oestrogen inhibited the induction of iNOS in murine macrophage cell line J774 [31]. The different findings in these studies could be due to the differences in the experimental conditions, such as the cell types used and/or stimuli employed.

In summary, the findings of our study demonstrate that oestrogen enhances host resistance to murine pulmonary MAC infection in vivo, possibly due to the enhancement by oestrogen of RNI production from activated macrophages. Human MAC pulmonary disease is usually refractory to chemotherapy. The findings of the present study in the murine system may shed light not only on the pathogenesis of the disease, but also on effective strategies for prevention and treatment of the disease.

Acknowledgments

This work was supported by a Grant-in-Aid (no. 10670542) for Scientific Research from the Ministry of Education of Japan and by a grant from the United States-Japan Cooperative Medical Science Program (Tuberculosis and Leprosy Section).

We thank Tokie Honma, Kazumi Kataoka, and Tomoko Ueda for technical assistance.

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