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The Journal of Immunology, 2004, 173: 1266-1275.
Copyright © 2004 by The American Association of Immunologists

Type I IFN Protects Permissive Macrophages from Legionella pneumophila Infection through an IFN-{gamma}-Independent Pathway1

Giovanna Schiavoni*, Claudia Mauri{ddagger}, Davide Carlei*, Filippo Belardelli*, Maddalena Castellani Pastoris{dagger} and Enrico Proietti2,*

Laboratories of * Virology and {dagger} Bacteriology and Medical Mycology, Istituto Superiore di Sanita’, Rome, Italy; and {ddagger} Center for Rheumatology Research, Department of Medicine, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-{gamma}. Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L. pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant. In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection. We demonstrate that treatment with type I IFN (IFN-{alpha}{beta}) totally inhibits the growth of L. pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages. IFN-{alpha}{beta} protective effect on permissive macrophages was comparable to that induced by IFN-{gamma}. Even low doses of either IFN-{alpha} or IFN-{beta} alone were effective in inhibiting L. pneumophila multiplication in macrophage cultures. Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-{alpha}{beta} significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-{alpha}{beta} in mediating the natural resistance of macrophages to L. pneumophila infection. Finally, addition of anti-IFN-{gamma}-neutralizing Ab did not restore Legionella growth in IFN-{alpha}- or IFN-{beta}-treated A/J or CD1 permissive macrophages, indicating that IFN-{alpha}{beta} effect was not mediated by IFN-{gamma}. This observation was further confirmed by the finding that IFN-{alpha}{beta} was effective in inhibiting L. pneumophila replication in macrophages from IFN-{gamma} receptor-deficient mice. Taken together, our results provide the first evidence for a role of IFN-{alpha}{beta} in the control of L. pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Legionella pneumophila is an intracellular bacterium that replicates in human alveolar macrophages and monocytes causing severe and often fatal pneumonia in immunocompromised individuals. The mechanisms by which L. pneumophila infection is controlled within the lung are still unclear, even though the ability of macrophages to suppress intracellular replication of the bacteria and to initiate adaptive immunity is thought to play a pivotal role in the outcome of legionellosis (1, 2). Immunostimulating Ags from L. pneumophila (LPS, heat shock proteins, and flagella) can induce macrophages to release a large number of inflammatory cytokines, such as IFN-{gamma}, TNF-{alpha}, IL-6, and IL-1, which are involved in the innate control of bacterial replication and can coordinate adaptive immune responses (3). In particular, IFN-{gamma} has been shown to restrict L. pneumophila replication by enhancing antimicrobial activity of macrophages. Furthermore, endogenous levels of IFN-{gamma} also appear to be critically involved in the resistance of macrophages to L. pneumophila infection (4, 5, 6, 7). In this regard, high permissiveness for L. pneumophila growth in thioglycolate (TG)3-elicited peritoneal macrophages from genetically deficient A/J mice is associated with the defective ability of these cells to produce IFN-{gamma} in response to infection (8). In contrast, peritoneal macrophages from many other strains of mice, such as C57BL/6, BALB/c, DBA/2, BDF-1, or C3H/HeN, are resistant to L. pneumophila infection both in vivo and in vitro (9). Such diversity in permissiveness to intracellular Legionella in inbred mice is known to be genetically controlled by a gene within the Lgn1 locus, which maps to chromosome 13 (10, 11, 12). Previous studies have shown that higher susceptibility of newborn, with respect to adults, CD1 mice to in vivo challenge with L. pneumophila correlated with increased permissiveness of peritoneal macrophages to bacteria replication, thus supporting the concept that immunomodulatory properties of macrophages are critical to confer resistance to L. pneumophila infection (13). Of note, it has been shown that CD1 peritoneal macrophages cultured for several days in vitro lose their natural resistance to infection with some viruses and that such increased susceptibility is due to lack of soluble autocrine and paracrine factors necessary for their natural resistance to virus infection, among which type I IFN (IFN-{alpha}{beta}) plays a central role (14, 15).

IFN-{alpha}{beta} is a family of cytokines expressed at low levels in virtually any cell type and inducible in high amounts during early phases of infection with viruses and other pathogens. IFN-{alpha}{beta} has been shown to exert a variety of immunomodulatory effects, including the enhancement of cytotoxic activity of NK and CD8+ T cells, the stimulation of dendritic cell differentiation and activity, and the enhancement of humoral responses (16, 17, 18, 19, 20). In macrophages, IFN-{alpha}{beta} can stimulate cytotoxicity and regulate the expression of many cytokines (21, 22). Importantly, low constitutive levels of endogenous IFN-{alpha}{beta} have been shown to be essential in conferring an antiviral state in macrophages (23). Besides their antiviral properties, IFN-{alpha}{beta} was found to promote antimicrobial activity against some intracellular microbial pathogens, such as Chlamydia spp., Toxoplasma gondii, Leishmania spp., or Mycobacterium avium (22, 24, 25, 26, 27). In particular, IFN-{alpha}{beta} was shown to be critically required for the release of autocrine IFN-{gamma} and the control of Chlamydia pneumoniae infection in bone marrow-macrophages (28). In contrast, IFN-{alpha}{beta} has been reported to counteract several IFN-{gamma}-mediated antimicrobial effects, such as inducible nitric oxide synthase and type II nitric oxide synthase (29, 30). These findings unravel heterogeneous effects of IFN-{alpha}{beta} on macrophages, implying that this cytokine may assume divergent functions in the control of intracellular bacteria.

In this study, we analyzed the role of IFN-{alpha}{beta} in the control of L. pneumophila infection in two models of permissive peritoneal macrophages: freshly isolated TG-macrophages from A/J mice, and in vitro aged TG-macrophages from CD1 mice. We demonstrate that IFN-{alpha}{beta} treatment strongly inhibits L. pneumophila replication in permissive macrophages and this effect is not mediated by IFN-{gamma}. We also show that endogenous levels of IFN-{alpha}{beta} can play an important role in conferring natural resistance to Legionella infection in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Five- to 8-wk-old male Swiss CD1, C57BL/6, and DBA/2 mice were obtained from Charles River Laboratories (Calco, Italy). Female A/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding pairs of IFN-{gamma}R–/– mice (strain 129/Sv-Ifngrtml) were obtained through the courtesy of Prof. I. Gresser (Institut National de la Santé et de la Recherche Médicale, Unité 255, Institut Curie, Paris, France). A colony was then bred and maintained at the Istituto Superiore di Sanità (Rome, Italy) under sterile conditions. All mice were treated in accordance with the European Union guidelines.

Bacteria

A virulent strain of L. pneumophila serogroup 6 was obtained from a fatal case of legionellosis in Italy.

IFN preparations

High titer mouse IFN-{alpha}{beta} was prepared in the mouse sarcoma C243-3 cell line following a method adapted from Tovey et al. (31). Briefly, suspension cultures of C243-3 cells infected with Newcastle disease virus were adjusted to pH 2.0 and kept at 0°C for 6 days. IFN-{alpha}{beta} was concentrated and partially purified by ammonium sulfate precipitation and dialysis against PBS. Purified mouse IFN-{alpha} and IFN-{beta} were obtained by sequential affinity chromatography on poly(U) and Ab-agarose columns, as described (32). Recombinant mouse IFN-{gamma} was kindly provided by Prof. W. Fiers (Ghent University, Ghent, Belgium).

Anti-IFN Abs

Ab to mouse IFN-{alpha}{beta} was prepared as previously described (33). The R4-6A2 rat:mouse hybridoma cell line (mAb to mouse IFN-{gamma}) was a generous gift from Dr. E. Havell (Trudeau Institute, Saranac Lake, NY). The ascites of mice injected with the hybridoma were partially purified by ammonium sulfate precipitation. The anti-mouse IFN-{gamma} had a neutralizing titer of 1/40 dilution against 8 IU of mouse IFN-{gamma}.

Culture of peritoneal macrophages

Resident macrophages were obtained from a peritoneal lavage with 5 ml of RPMI 1640 medium supplemented with 2% FCS (Invitrogen Life Technologies, Grand Island, NY). Elicited macrophages were harvested 3 days after i.p. injection of 3 ml of 3% TG (Difco, Livonia, MI). Peritoneal exudate cells were washed, resuspended in RPMI 1640 medium supplemented with 10% FCS, 1% penicillin, 1% streptomycin, and incubated in 96 flat-bottom plates (106 cells per well in 0.1 ml) for 3 h at 37°C. Nonadherent cells were then removed by vigorous washing (three times), and macrophages monolayers were incubated for 1–10 days in antibiotic-free RPMI containing 10% FCS. Fresh medium was given every 3 days. Unless otherwise specified, treatments with cytokines and/or Abs to IFNs were performed 18 h before and immediately after Legionella infection (see Results).

Infection and enumeration of L. pneumophila

If not otherwise stated, macrophage monolayers (~106 cells per well, in 96 flat-bottom plates) were infected with 5 x 104 bacteria per well (0.05 multiplicity of infection (m.o.i.)). Infection was performed as follows: cell monolayers were washed and infected in triplicate with L. pneumophila for 2 h at 37°C. Macrophages were then washed three times to remove nonphagocytized bacteria and further incubated in 0.1 ml of antibiotic-free medium for various times. The starting point of this incubation represents the zero time. Cell monolayers were lysed with 0.1 ml of sterile distilled water. The number of viable bacteria in culture lysates (zero time) or in the combined culture lysates and culture supernatants was determined by standard plate counts. Culture lysates and the combined mixtures were appropriately diluted in sterile water, plated on buffered charcoal-yeast extract agar (Difco), incubated for 4 days, and then counted for CFU.

ELISA

Supernatants from either uninfected or Legionella-infected macrophages were assayed for the presence of TNF-{alpha} or IFN-{gamma} using immunoenzimatic ELISA kits (R&D Systems, Minneapolis, MN).

Statistical analysis

Data were analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro-aged CD1 TG-macrophages are permissive to L. pneumophila growth

Resident peritoneal macrophages from A/J mice are known to be resistant to L. pneumophila infection, but they become highly permissive when elicited with TG in vivo (9). These observations were confirmed in our preliminary experiments (Fig. 1A).



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  		FIGURE 1. L. pneumophila growth in permissive macrophages in vitro. A, Peritoneal macrophages from either untreated or TG-treated A/J mice were cultured in 96-well plates for 18 h and then infected with L. pneumophila at 0.05 m.o.i. for 2 h. Cells were then washed and either lysed (day 0) or further incubated for the indicated time points. B, TG-elicited peritoneal macrophages from CD1 mice were cultured for 1, 7, or 10 days, then infected with L. pneumophila as in A. C, TG-elicited peritoneal macrophages from DBA/2 and C57BL/6 mice were cultured for 1 or 7 days, and then infected with L. pneumophila. Bacterial load in macrophage monolayers was determined as indicated in Materials and Methods. Data represent the mean CFU ± SD of triplicate cultures (*, p ≤ 0,001; **, p ≤ 0,05). Representative results of one of at least three separate experiments are shown.

 
Unlike A/J mice, TG-elicited macrophages from CD1 mice are resistant to L. pneumophila infection in vitro (13). As previous findings showed that in vitro "aging" of CD1 macrophages renders these cells highly susceptible to infection with certain viruses (14), we asked whether these aged CD1 macrophages could also become permissive to L. pneumophila growth. To this purpose, peritoneal macrophages from TG-treated CD1 mice were kept in culture for 7–10 days and subsequently infected with L. pneumophila. Control macrophage cultures consisted of freshly isolated cells that had been left in culture for 18 h. After a 2 h infection, all cell monolayers were extensively washed in warm medium and further cultured for various times (0, 3, 6, and 8 days) and then lysed to measure bacterial load. As expected, fresh macrophage cultures were poorly permissive to L. pneumophila replication. Conversely, in vitro-aged macrophages displayed significantly higher susceptibility to bacteria growth, with 10-fold increase for 7-day-aged and 600-fold increase for 10-day-aged macrophages in CFU numbers at day 3 postinfection, with respect to fresh cultures (Fig. 1B). However, 10-day-aged cell monolayers lost the capacity to adhere to culture plates (not shown), therefore compromising optimal determination of bacteria:macrophages ratios. For this reason, 7-day aged macrophages were routinely used for the subsequent experiments.

We then asked whether the increased susceptibility to L. pneumophila infection of TG-macrophages upon extended culture was an exclusive feature of CD1 mice or a more general phenomenon in mice. To this end, we analyzed L. pneumophila growth in aged TG-macrophages from other strains of mice that are known to be naturally resistant to Legionella intracellular growth. As illustrated in Fig. 1C, 7-day-aged peritoneal TG-macrophages from C57BL/6 and DBA/2 mice displayed a significant increase in their permissiveness to L. pneumophila replication as compared with fresh cultures, with a 5- and 16-fold increase, respectively, in CFU numbers at 3 days after infection.

IFN-{alpha}{beta} inhibits L. pneumophila replication in permissive macrophages

We investigated whether IFN-{alpha}{beta} could play a role in the control of L. pneumophila growth in the two models of permissive macrophages: fresh A/J and 7-day-aged CD1 TG-elicited peritoneal macrophages. To this end, fresh A/J or 7-day-aged CD1 cell monolayers were cultured for 18 h in the presence or absence of IFN-{alpha}{beta} or IFN-{gamma}. Macrophages were then infected with L. pneumophila for 2 h, washed, and either lysed immediately thereafter or after an incubation period of 3–6 days in the presence or absence of the various cytokines. As shown in Fig. 2, IFN-{alpha}{beta} treatment resulted in a marked inhibition of L. pneumophila replication in both fresh A/J and aged CD1 macrophages. In fact, at day 3, the CFU numbers significantly decreased with respect to the initial uptake (day 0) and virtually no bacteria (≤10 CFU) could be detected at day 6 after infection in IFN-{alpha}{beta}-treated A/J (Fig. 2A) or CD1 (Fig. 2B) macrophage cultures as compared with untreated cells. Notably, IFN-{alpha}{beta} inhibitory effect on Legionella growth was comparable with that induced by IFN-{gamma} in both A/J and CD1 macrophage models. No significant differences in CFU numbers were observed in all macrophage cultures at day 0, suggesting that neither cytokines affected the initial bacteria uptake.



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  		FIGURE 2. IFN-{alpha}{beta} inhibits the growth of L. pneumophila in permissive macrophages. Freshly isolated A/J (A) and 7-day-cultured CD1 (B) TG-elicited macrophages were incubated in the presence of IFN-{alpha}{beta} (1000 U/ml) or IFN-{gamma} (100 U/ml) for 18 h at 37°C. Cell monolayers were then infected with L. pneumophila (0.05 m.o.i.) for 2 h, washed, and then incubated for various times in fresh medium containing or not containing the cytokines. Each point represents the mean CFU ± SD of triplicate macrophage cultures. Data are representative of one of five experiments.

 
To test whether the protective effect of IFN-{alpha}{beta} on infected macrophages was due to a preferential action of either IFN-{alpha} or IFN-{beta} subtypes, we treated permissive macrophages with IFN-{alpha}, or IFN-{beta} alone or in combination, and compared their effect with that mediated by partially purified IFN-{alpha}{beta}. Either IFN-{alpha} or IFN-{beta} displayed a similar inhibitory effect on L. pneumophila growth in aged CD1 macrophages (Fig. 3A), as well as in A/J cultures (data not shown), as revealed by CFU determination at days 3 and 6 after infection. No significant differences in CFU numbers were observed in cultures treated with single IFN subtypes, with respect to those exposed to partially purified IFN-{alpha}{beta} or to the combination of IFN-{alpha} plus IFN-{beta} (Fig. 3A). In addition, the inhibitory effect of both IFN-{alpha} and IFN-{beta} on L. pneumophila growth in permissive CD1 macrophages was dose-dependent (Fig. 3B). IFN-{beta} appeared to be more efficient than IFN-{alpha} in inhibiting bacterial replication. Notably, even at doses as low as 0.6 U/ml, both IFN-{alpha} and IFN-{beta} were effective in restricting bacteria replication, as revealed by a 5-fold reduction in CFU number at day 3 with respect to untreated cultures (Fig. 3B).



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  		FIGURE 3. Effect of IFN-{alpha} or IFN-{beta} on L. pneumophila growth in CD1 macrophages. Seven-day-aged, TG-elicited peritoneal macrophages from CD1 mice were treated with different preparations of mouse type I IFN: IFN-{alpha} (400 U/ml), IFN-{beta} (400 U/ml), a combination of IFN-{alpha} and IFN-{beta}, or partially purified IFN-{alpha}{beta} (1000 U/ml; A) and IFN-{alpha} or IFN-{beta} in various amounts (B). Cells were incubated for 18 h at 37°C and then infected with L. pneumophila. Two hours later, macrophages were washed and further incubated in the presence or absence of the various cytokines. Bacterial growth was detected at the indicated time points and is expressed as CFU ± SD of triplicate macrophage cultures. Representative data of one of five experiments are shown.

 
Previous studies evidenced that IFN-{alpha}{beta} can promote the differentiation of monocytes as well as macrophages (34, 35). Therefore, we asked whether the protective effect mediated by type I IFN was a result of a direct effect on the antibacterial activity of macrophages or was exclusively due to stimulation of macrophage differentiation. In the first case, a single IFN treatment after Legionella infection would be sufficient to determine growth inhibition. Thus, macrophage cultures from A/J and CD1 mice were exposed to IFN-{alpha} once after infection with L. pneumophila. As shown in Fig. 4A, a single postinfection treatment with IFN-{alpha} induced significant reduction of bacteria replication in susceptible A/J (200-fold), as well as in CD1 (100-fold), macrophages, with respect to control untreated cultures. However, the greatest inhibition of bacteria growth was observed when cell monolayers had been preincubated with IFN-{alpha} before infection. Similarly, a single exposure to IFN-{beta} after infection resulted in a decreased multiplication of L. pneumophila in A/J macrophages as compared with untreated cells, even though complete growth inhibition occurred only when IFN-{beta} was administered twice (Fig. 4B).



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  		FIGURE 4. Effect of single exposure to IFN-{alpha} or IFN-{beta} after L. pneumophila infection. A, TG-elicited fresh A/J or 7-day-aged CD1 macrophages were incubated for 18 h in the presence or absence of IFN-{alpha} (200 U/ml). Cells were then infected with L. pneumophila for 2 h, washed, and then incubated for 3 days with or without IFN-{alpha}, as indicated in the figure. Each bar represents the mean CFU ± SD of triplicate macrophage cultures. B, Fresh A/J TG-macrophages were incubated alone or in the presence of IFN-{beta} (200 U/ml) for 18 h and then infected with L. pneumophila. After 2 h, cells were washed and incubated for 3–6 days with or without cytokines, as indicated in the figure. Each point represents the mean CFU ± SD of triplicate macrophage cultures. Data are representative of three separate experiments.

 
IFN-{alpha}{beta} Ab increases the susceptibility of macrophages to L. pneumophila infection

The finding that low doses of IFN-{alpha}{beta} are sufficient to confer resistance to L. pneumophila infection raised the question of whether endogenous levels of IFN-{alpha}{beta} could play a role in the natural resistance of macrophages to Legionella. To test this, we treated fresh cultures from CD1 mice, which are resistant to L. pneumophila infection, with anti-IFN-{alpha}{beta} Ab for 18 h, then challenged them for 2 h with L. pneumophila (at various m.o.i.). Treatment with anti-IFN-{alpha}{beta} Ab was repeated after infection. As illustrated in Table I, bacteria growth was poorly supported in untreated fresh macrophage cultures. Conversely, anti-IFN-{alpha}{beta} treatment markedly enhanced the ability of macrophages to replicate L. pneumophila, as revealed by increased CFU numbers at day 1 (4- to 12-fold) and at day 2 (100- to 1000-fold) after infection, with respect to control macrophages (Table I).


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  		Table I. Effect of anti-IFN-{alpha}{beta} Ab on L. pneumophila replication in freshly isolated TG-elicited peritoneal macrophages from CD1 mice

 
IFN-{alpha}{beta} inhibitory effect on L. pneumophila is independent on IFN-{gamma}

IFN-{gamma} is released by macrophages early during L. pneumophila infection and plays a key role in the control of bacteria growth (4, 5, 6, 7, 8). Therefore, we asked whether the protective action of IFN-{alpha}{beta} on permissive macrophages was mediated, at least in part by IFN-{gamma}. First, we measured the release of IFN-{gamma} in supernatants from macrophages under different culture conditions, upon L. pneumophila infection and in comparison to bacterial growth. Fig. 5A shows the levels of IFN-{gamma}, 3 days after infection with L. pneumophila, in fresh and aged CD1 macrophages, the latter treated or not with IFN-{alpha}. Bacterial infection could induce IFN-{gamma} only in fresh macrophage cultures. Conversely, aged untreated macrophages were unable to release this cytokine after the Legionella challenge. Accordingly, L. pneumophila replication was reduced in fresh macrophages, whereas high bacterial load was found in aged cultures (Fig. 5B). IFN-{alpha} treatment did not restore IFN-{gamma} release in aged macrophages after infection with L. pneumophila (Fig. 5A), although it could efficiently block bacteria replication, as detected 3 days after infection (Fig. 5B). In untreated macrophage cultures from A/J mice, IFN-{gamma} was not released upon L. pneumophila infection (Fig. 5C), confirming previous findings (8). Conversely, IFN-{alpha} treatment induced IFN-{gamma} production in A/J cell monolayers exposed to Legionella (Fig. 5C). In parallel, bacteria replication was totally inhibited in IFN-{alpha}-treated macrophages, whereas high numbers of CFU were found in untreated cultures (Fig. 5D).



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  		FIGURE 5. IFN-{gamma} production in cultures of peritoneal macrophages. A and B, TG-elicited peritoneal macrophages from CD1 mice were cultured for either 1 or 7 days. Seven-day macrophages were then cultured for an additional 18 h in the presence or absence of IFN-{alpha} (200 U/ml). Cell monolayers were then infected with L. pneumophila (0.05 m.o.i.) for 2 h at 37°C, washed, and further incubated in the presence or absence of IFN-{alpha}. Three days after infection, the levels of IFN-{gamma} were measured in supernatants from cell cultures by ELISA (A). Intracellular L. pneumophila in the same cultures was determined by lysis in water (B). IFN-{gamma} levels (C) and bacterial growth (D) in freshly isolated A/J macrophages treated or not with IFN-{alpha}, were detected at 3 days after infection with L. pneumophila. IFN-{gamma} levels were determined in pooled triplicate cultures. Legionella load is represented as CFU ± SD of triplicate macrophage cultures. One representative experiment of three separate ones is shown.

 
The above data suggested that, in the case of A/J permissive macrophages, blocking of L. pneumophila replication by IFN-{alpha} correlated with induction of IFN-{gamma} release, raising the possibility that in this mouse model IFN-{alpha} action could be mediated by IFN-{gamma}. To test this hypothesis, we treated aged CD1 and fresh A/J macrophages with IFN-{alpha} or IFN-{beta} in presence or absence of anti-IFN-{gamma} neutralizing Ab, 18 h before and immediately after infection with L. pneumophila. As illustrated in Fig. 6A, treatment with anti-IFN-{gamma} Ab did not restore Legionella growth in CD1 macrophage cultures exposed to either subtypes of type I IFN. Of interest, CD1 macrophages treated with anti-IFN-{gamma} showed a significant increase in bacteria load, as revealed by higher CFU counts at day 3 (5-fold) and day 6 (3-fold) with respect to untreated cells. Similarly, addition of anti-IFN-{gamma} Ab to fresh A/J macrophages did not block IFN-{alpha} (not shown) or IFN-{beta}-induced inhibition of L. pneumophila growth (Fig. 6B). To confirm that IFN-{alpha}{beta} effect was independent on IFN-{gamma} signals, we used peritoneal TG-macrophages from IFN-{gamma}R–/– mice. As illustrated in Fig. 6C, fresh macrophage cultures from these mice were highly permissive to L. pneumophila replication in vitro at 3 and 6 days after infection. Notably, treatment with IFN-{beta} was completely effective in inhibiting bacteria multiplication in IFN-{gamma}R–/– macrophages (Fig. 6C).



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  		FIGURE 6. IFN-{alpha}{beta} inhibits L. pneumophila intracellular growth in an IFN-{gamma}-independent manner. A, TG-elicited, 7-day-aged CD1 macrophages were incubated for 18 h with IFN-{alpha} (200 U/ml), IFN-{beta} (200 U/ml), either alone or in combination with anti-IFN-{gamma}-neutralizing Ab (400 U/ml), then infected with L. pneumophila. Two hours after infection, cells were washed and reincubated for various times in the presence of the various cytokine and Ab combinations. B, Freshly isolated A/J were cultured alone or in the presence of IFN-{beta} (200 U/ml) with or without anti-IFN-{gamma} Ab (400 U/ml) for 18 h and then infected. C, Freshly isolated TG-elicited macrophages from IFN-{gamma}R–/– mice were cultured for 18 h alone or in the presence of IFN-{beta} (200 U/ml), and then infected with L. pneumophila. At the indicated time points, cells were lysed with water to assess for Legionella multiplication. Bacteria counts are expressed as CFU ± SD of triplicate macrophage cultures. One representative experiment of two is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that both IFN-{alpha} and IFN-{beta} play a key immunomodulatory role in the resolution of L. pneumophila replication in two mouse models of permissive peritoneal macrophages: fresh cultures from A/J mice and in vitro-aged cells from CD1 mice. TG-treated A/J mice have been shown to be more susceptible to L. pneumophila infection in vivo, with respect to other strains (36). Furthermore, TG-macrophages from A/J, unlike most of other mouse strains, are highly permissive to L. pneumophila intracellular growth in vitro (9). Although the molecular basis regulating host resistance to L. pneumophila infection has been associated to the Lgn1 locus, with resistance being fully dominant over susceptibility, the immunological mechanisms responsible for the higher susceptibility of A/J macrophages to L. pneumophila infection are still unclear. It has been observed that A/J macrophages are defective in IFN-{gamma} induction after Legionella infection, in a process that likely involves the inhibition of macrophage-derived IL-12 by L. pneumophila, because treatment with exogenous IL-12 restores both IFN-{gamma} release and resistance to Legionella infection in A/J macrophages (8, 37). In our study, we show that, even though IFN-{gamma} production by A/J macrophages infected with L. pneumophila can also be induced by IFN-{alpha}{beta} treatment, this endogenous IFN-{gamma} is clearly not required for IFN-{alpha}{beta}-mediated resolution of replicative bacteria, because such process was not affected by treatment with anti-IFN-{gamma} Ab. This finding suggests that L. pneumophila growth in AJ macrophages can be controlled through IFN-{gamma}-independent pathways. Consistent with this hypothesis, it has been described that A/J macrophages activated by LPS in vivo not only become resistant to subsequent L. pneumophila infection in vitro, but can also confer resistance to cocultured permissive A/J macrophages (38). Although the authors were not able to identify the factors mediating such transfer of nonpermissiveness, it was shown that neither IFN-{gamma}, IL-1, IL-6, nor TNF-{alpha} was involved. In this regard, it is worth mentioning that expression of type I IFN, especially IFN-{beta}, is also enhanced by LPS in macrophages and is correlated with some LPS-induced antimicrobial functions, such as NO generation (15, 39, 40). The finding that IFN-{alpha}{beta} can effectively block L. pneumophila growth in A/J macrophage cultures is in contrast with a previous work showing that IFN-{alpha} had no effect in inhibiting Legionella replication in these cells (41). This discrepancy might be partly explained by the fact that the authors of this study used recombinant IFNs which may possess different biological activity with respect to the natural IFN preparations used in our study. The different IFN preparations may induce different kinetics of IFN-dependent effects. We may envisage that the authors have failed to detect an inhibitory effect because they observed L. pneumophila growth only on days 1 and 2 after infection. In contrast, our findings are in agreement with a study showing that IFN-{beta} exhibit a suppressive effect on L. pneumophila intracellular replication in vitamin D3-treated HL-60 cells, a human cell line displaying monocyte-like morphology (42).

As an alternative approach to investigate the role of IFN-{alpha}{beta} in L. pneumophila infection, we used in vitro-aged CD1 macrophages. We show that TG-elicited CD1 macrophages, as well as those from the inbred C57BL/6 and DBA/2 strains, that are normally resistant to L. pneumophila infection, become permissive after 7 days of culture. This result was somehow expected, because previous studies demonstrated that in vitro aging of CD1 macrophages results in increased susceptibility to viral infection (14). In the case of antiviral resistance, it was shown that lack of exposure to paracrine and autocrine IFN-{alpha}{beta} was the mechanism responsible for the acquired permissiveness to infection in macrophages (14, 15, 23, 43). Although the mechanisms mediating the loss of resistance to L. pneumophila infection in aged CD1 macrophages are unknown, evidence provided here suggests that IFN-{alpha}{beta} may be involved. First, the finding that treatment with exogenous IFN-{alpha}{beta} even at very low doses can effectively inhibit L. pneumophila replication in permissive macrophages indicates that low levels of IFN-{alpha}{beta}, comparable to spontaneous basal expression, might be sufficient to protect macrophages from infection. Second, the observation that administration of anti-IFN-{alpha}{beta} Ab to fresh CD1 monolayers results in a loss of their natural resistance to L. pneumophila infection implies that endogenous IFN-{alpha}{beta} plays an important role in protecting macrophages from bacteria challenge. In this respect, previous work evidenced that autocrine IFN-{alpha}{beta} is required for the enhancement of macrophage phagocytosis activated by M-CSF and IL-4 (44).

IFN-{gamma} was also efficient in protecting aged CD1 macrophages from L. pneumophila infection, thus confirming the pivotal role played by this cytokine in the control of L. pneumophila replication. Interestingly, addition of anti-IFN-{gamma} to these cells further increased their ability to support bacterial growth, suggesting that autocrine IFN-{gamma} is also important in conferring resistance to infection in aged macrophages (6, 7). Consistent with this, we found that IFN-{gamma} was present in supernatants of aged cells before infection even in higher amounts with respect to those found in fresh macrophage cultures (data not shown). In this regard, IFN-{gamma} release by macrophages has been described to increase with the time of culture, as a result of a positive feedback loop mediated by IFN-{gamma} on its own gene (45). Nevertheless, IFN-{gamma} was not induced upon infection in aged macrophages, unlike in fresh cultures, and IFN-{alpha}{beta} treatment did not restore IFN-{gamma} production in aged cells. Accordingly, blocking of IFN-{gamma} activity did not revert IFN-{alpha}{beta}-induced protective effect on CD1 aged macrophages infected with L. pneumophila, again indicating that IFN-{alpha}{beta} action was not mediated by IFN-{gamma}. The concept that IFN-{alpha}{beta} inhibitory action on L. pneumophila replication in permissive macrophages is independent on IFN-{gamma} was even further confirmed by the finding that IFN-{alpha}{beta} was effective in blocking bacterial growth in macrophages from IFN-{gamma}R–/– mice. Expectedly, these cells displayed high susceptibility to L. pneumophila replication due to lack of autocrine IFN-{gamma} (6, 7). However, these cells became highly restrictive to L. pneumophila growth upon IFN-{beta} treatment. These results suggest that, regardless of the immunological defect causing permissiveness in macrophages, the protective effect induced by IFN-{alpha}{beta} against L. pneumophila infection was triggered through a pathway independent on IFN-{gamma} signals. Notably, IFN-{alpha}{beta} did not interfere with IFN-{gamma}-mediated resolution of replicative L. pneumophila, because coadministration of both cytokines to either CD1 or A/J permissive macrophages resulted in effective inhibition of L. pneumophila replication (data not shown).

At present, the mechanisms by which IFN-{alpha}{beta} inhibits L. pneumophila intracellular growth in macrophages are unknown. The finding that IFN-{alpha}{beta} can partly restrict replicative bacteria when administered after infection with L. pneumophila suggests that this cytokine may stimulate antimicrobial activity of macrophages. In this regard, evidence for the IFN-{beta}-promoting effect on macrophages antibacterial function includes the stimulation of NO secretion and the deprivation of intracellular iron through down-regulation of the transferrin receptor (21, 39, 40, 46). Accordingly, iron deprivation seems one of the mechanisms involved in both LPS and IFN-{gamma}-induced inhibition of intracellular proliferation of L. pneumophila in A/J macrophages (47, 48). Nevertheless, we observed complete resolution of replicative L. pneumophila only when IFN-{alpha}{beta} was given both prior and after infection, thus indicating that IFN-{alpha}{beta} immunomodulatory role in priming macrophage differentiation and activation is a critical process resulting in total protection against forthcoming infection. In this respect, type I IFN may prime macrophages affecting the early events of Legionella infection, by mechanisms layered onto the Lgn1 locus, which is thought to influence Legionella internalization at the phagocytic level (49). Alternatively, IFN-{alpha}{beta} may regulate the secretion of other cytokines, which in turn can control L. pneumophila replication. However, one such possible candidate cytokine, TNF-{alpha}, which is induced by L. pneumophila and is known to control bacterial replication in macrophages (50, 51), was not found to be affected by IFN-{alpha}{beta} in our macrophage cultures (data not shown).

Further studies will be needed to elucidate the IFN-{alpha}{beta}-induced immunomodulatory changes on macrophages which are responsible for the control of L. pneumophila infection. Regardless of the mechanism involved, we suggest that both autocrine and paracrine IFN-{alpha}{beta} play a critical immunomodulatory role in stimulating macrophage differentiation and/or activation, which maintains these cells in a status of resistance to infection, resulting in a prompt resolution of intracellular L. pneumophila. On the whole, our results provide strong evidence for the role of type I IFN on the control of L. pneumophila infection and disclose wider perspectives for cytokine-based potentiation of antibacterial therapies.


   Acknowledgments
 
We thank Drs. Martin E. Rottenberg, Patrizia Puddu, and Fabrizio Mattei for critical review of the manuscript and helpful comments. We are also thankful to Annamaria Fattapposta and Angela Fresolone for excellent secretarial assistance.


   Footnotes
 
1 This work was supported by the European Community ("Experimental and clinical dissection of innate immunity against intracellular pathogens" contract Bio QLK2-CY-2002-0846). Back

2 Address correspondence and reprint requests to Dr. Enrico Proietti, Laboratory of Virology, Istituto Superiore di Sanita’, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: proietti{at}iss.it Back

3 Abbreviations used in this paper: TG, thioglycolate; m.o.i., multiplicity of infection. Back

Received for publication November 20, 2003. Accepted for publication May 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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