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The Journal of Immunology, 2001, 167: 375-383.
Copyright © 2001 by The American Association of Immunologists

Enhanced Protection Against Fatal Mycobacterial Infection in SCID Beige Mice by Reshaping Innate Immunity with IFN-{gamma} Transgene1

Zhou Xing2, Anna Zganiacz, Jun Wang and Sudhir K. Sharma

Department of Pathology and Molecular Medicine, and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Humans with immune-compromised conditions such as SCID are unable to control infection caused by normally nonpathogenic intracellular pathogens such as Mycobacterium bovis bacillus Calmette-Guérin. We found that SCID beige mice lacking both lymphocytes and NK cells had functionally normal lung macrophages and yet a selectively impaired response of type 1 cytokines IFN-{gamma} and IL-12, but not TNF-{alpha}, during M. bovis bacillus Calmette-Guérin infection. These mice succumbed to such infection. A repeated lung gene transfer strategy was designed to reconstitute IFN-{gamma} in the lung, which allowed investigation of whether adequate activation of innate macrophages could enhance host defense in the complete absence of lymphocytes. IFN-{gamma} transgene-based treatment was initiated 10 days after the establishment of mycobacterial infection and led to increased levels of both IFN-{gamma} and IL-12, but not TNF-{alpha}, in the lung. Lung macrophages were activated to express increased MHC molecules, type 1 cytokines and NO, and increased phagocytic and mycobactericidal activities. Activation of innate immunity markedly inhibited otherwise uncontrollable growth of mycobacteria and prolonged the survival of infected SCID hosts. Thus, our study proposes a cytokine transgene-based therapeutic modality to enhance host defense in immune-compromised hosts against intracellular bacterial infection, and suggests a central effector activity played by IFN-{gamma}-activated macrophages in antimycobacterial cell-mediated immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Host defense against intracellular infections of macrophages, including mycobacterial infection, requires the type 1 cell-mediated immunity (1, 2). Activation of both type 1 T cells and macrophages is believed to be essential to effective control of intracellular infection. The generation of type 1 T cells is facilitated by cytokine IL-12. Both activated type 1 CD4 and CD8 T cells as well as NK cells may contribute to host defense by secreting macrophage-activating type 1 cytokines, including IFN-{gamma} and TNF-{alpha} (1, 2, 3). In vitro studies suggest that macrophages activated by these cytokines acquire an increased ability to control the replication of intracellular mycobacteria (3). In addition to activated macrophages, however, both CD4/CD8 T cells and NK cells are also capable of antimycobacterial effector activities by directly killing infected macrophages, inhibiting mycobacterial replication, and lysing mycobacteria (3, 4, 5, 6). Thus, the issue regarding to which extent activated macrophages by themselves are able to control mycobacterial infection in vivo has remained to be understood.

Humans with such immune-compromised conditions as SCID, AIDS, and genetic deficiencies in type 1 cytokines IFN-{gamma} and IL-12 or their receptors are especially susceptible to intracellular infection by normally otherwise nonpathogenic strains of mycobacteria such as Mycobacterium bovis bacillus Calmette-Guérin (BCG)3 (1, 2, 3, 7, 8). It is known that chemotherapeutic modalities are often ineffective in these hosts without the aid provided by key intrinsic immune components. Future effective therapy will most likely involve the use of immunotherapeutics aiming to enhance the function of remaining immune components such as macrophages in these hosts. However, the rationale and feasibility of such strategy still remain to be established. Apparently, the development of this strategy rests on the important definition of the ability of macrophages to control mycobacterial infection in vivo, independent of cytotoxic T and NK cells.

Since the lung is naturally much more prone to mycobacterial infection than other tissue sites (9, 10), we employed a SCID beige mouse model of pulmonary M. bovis BCG infection that we have previously established (11) to investigate several questions of fundamental importance: 1) whether SCID hosts have an intact tissue macrophage population available for immune activation; 2) whether SCID hosts have impaired type 1 cytokine responses during myocbacterial infection; 3) whether a cytokine transgene-based therapeutic modality aiming to activate innate macrophages, when given after the establishment of infection, is able to enhance the control of mycobacterial replication in the absence of both lymphocytes and NK cells; and 4) if so, what are the mechanisms.


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

C.B-17 SCID beige mice were bred in our central animal facility under Level A specific pathogen-free housing conditions until use. Immune-competent BALB/c and C57BL/6 mice (Harlan, Indianapolis, IN) were housed under Level B specific pathogen-free housing conditions until use. After infection, all mice were kept in autoclaved cages with autoclaved bedding, food, water, and microfilter lids in a biohazard Level B facility. All experiments performed were in accordance with the guidelines of the Animal Research Ethics Board of McMaster University.

Infectious agent and viral cytokine gene transfer vectors

Live M. bovis BCG was originally obtained from Connaught Laboratories (North York, Ontario, Canada). It was grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with Middlebrook OADC enrichment (Life Technologies, Gaithersburg, MD), 0.002% glycerol, and 0.05% Tween 80. A replication-deficient adenoviral vector was used for cytokine gene transfer in vivo. This virus has its E1 and E3 genomic regions partially deleted, but remains fully infectious (12). As control, a replication-deficient adenoviral vector that does not contain cytokine transgene was used. All adenoviral gene vectors were amplified, purified, and titrated in our Center by using the standard procedures previously described by us (12).

Pulmonary mycobacterial infection and intranasal cytokine gene transfer

Pulmonary mycobacterial infection was established via the airway, as previously described (11, 13, 14). Before infection, BCG stock solution was diluted in PBS, and the preparation was sonicated to ensure proper dispersion of mycobacteria. Mice were infected by intratracheal instillation of live BCG at a dose of 5 x 105 CFU in a total volume of 40 µl/mouse, as previously described. Recombinant replication-deficient adenoviral vector expressing murine IFN-{gamma} (15) (kindly provided by J. Kolls at Louisiana State University, New Orleans, LA) or murine GM-CSF (16) or the control vector (Add170) was diluted into 4 x 108 PFU in a total volume of 30 µl in PBS. Intrapulmonary cytokine gene transfer was conducted by intranasal (i.n.) delivery of adenoviral vector, as previously described (16). Briefly, half of 30 µl virus suspension was applied to the mouse nasal orifice so that the mouse could breathe in the suspension naturally. The second half was applied in the same way thereafter. We have previously demonstrated that after i.n. gene transfer, transgene was expressed primarily by airway epithelial cells for a period of 10–12 days (16, 17, 18).

Bronchoalveolar lavage, lung histology, and lung macrophages isolation

At selected time points postintrapulmonary gene transfer, groups of mice were sacrificed and lungs were removed and subjected to bronchoalveolar lavage (BAL), followed by perfusion with 10% Formalin for histology. BAL was conducted by following a standard procedure previously described (11, 13). Briefly, to collect BAL fluid, a polyethylene tube (Becton Dickinson, Sparks, MD) was used to cannulate the trachea. Lungs were lavaged twice with PBS. BAL samples were spun at 4000 rpm for 2 min at 4°C, and supernatants were removed and stored at -20°C until cytokine assay. Lungs were fixed in 10% Formalin by perfusion. Both left and right lungs were sectioned from top to bottom, resulting in four to five cross-sectional pieces of tissue from each side. Tissues were then embedded in paraffin, cut into 4- to 5-µm sections, and stained with H&E or Ziehl-Neelsen for identification of mycobacteria.

Following BAL, alveolar macrophages were collected. For each experiment, macrophages isolated from three to five mice were pooled. These cells were resuspended in RPMI culture media containing 10% FBS and cultured for 3 days in 96-well plates at a density of 0.1 x 106 cells/well without or with various stimuli. The supernatants were collected and stored at -20°C until cytokine assay.

Measurement of cytokines in BAL and macrophage culture supernatants

Cytokines were measured in BAL and cell culture supernatants by specific ELISA. All ELISA kits were purchased from either R&D Systems (Minneapolis, MN) (IFN-{gamma} and TNF-{alpha}) or Biosource (Montréal, Québec, Canada) (IL-12). The sensitivity of detection for all of these ELISA kits was <=5–10 pg/ml.

Mycobacterial colony enumeration assay

A colony assay was performed as previously described (13). Briefly, lungs were placed in 4.5 ml PBS/0.05% Tween 80, cut into small pieces (2–3 mm) under sterile conditions, and homogenized with a tissue homogenizer. Homogenates were allowed to settle on ice for 30 min, and properly diluted homogenates were plated onto each plate of Middlebrook 7H10 agar containing OADC enrichment (Difco). Plates were incubated at 37°C, and colonies were counted using a dissecting microscope at days 11 to13.

Measurement of NO production

The release of NO by lung macrophages was determined by measuring the concentration in culture supernatants of the end product of NO, nitrite, as previously described (19). Briefly, diluted supernatants were mixed at a 1:1 ratio with Griess reagent buffer (Sigma, St. Louis, MO). The absorbance was determined at 540 nm by using a spectrophotometer. The final concentrations of nitrite were calculated from a standard curve derived from prepared solutions of NaNO2 of known concentrations.

Evaluation of macrophage activation by FACS analysis

Immunostaining and FACS analysis procedures were conducted as previously described (18, 20). Briefly, isolated total leukocytes from SCID mouse lung were incubated with anti-FcR Ab 2.4G2 on ice for 15 min and then stained with FITC-labeled anti-MHC class I or biotinylated anti-MHC class II Abs on ice for 30 min. After wash with PBS/0.2% BSA, the cells were labeled with PE-conjugated anti-mouse CD11b (Mac-1) Ab for 30 min on ice. Data were collected from FACScan (Becton Dickinson, Sunnyvale, CA). Analysis was performed by gating on the macrophage-rich area, excluding lymphocytes and dead cells. Since this area might also contain some granulocytes, the results from FACS analysis were interpreted by taking into account the percentage of macrophages in total BAL-derived leukocytes determined by differential staining of cytospins of BAL cells before FACS staining.

Macrophage phagocytosis and mycobactericidal assays

Macrophages were isolated from the lung of naive SCID mice receiving the control or IFN-{gamma} gene transfer vector. Cells were allowed to interact with live BCG bacilli (1:10 macrophage:bacilli) for 2 h at 37°C in 24-well plates at a density of 0.2 x 106 cells/well in RPMI media containing 5% FBS without antibiotics. Free, nonphagocytosed bacilli were removed by rinsing the monolayer of macrophages three to four times with culture media. Macrophages were then removed from the plate, and cell cytospins were made. Cytospins were stained with cold KINYOUN stain for acid-fast bacilli. At least 200–300 cells were counted on each cytospin. Both the number of intracellular bacilli in each macrophage and the number of macrophages that phagocytosed bacilli were recorded. To determine the rate of intracellular mycobacterial replication, macrophages were cultured with live BCG bacilli, as above. At both days 0 and 3, 0.5 ml cold 0.25% SDS was introduced into the plate to lyse macrophages. The number of intracellular bacilli was compared between day 0 and day 3 by a colony enumeration assay.

Data analysis

Whenever applicable, the difference comparison was made by using a Student t test. The difference was considered statistically significant when p <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of macrophages in the lung of naive SCID mice

We have previously reported that SCID beige hosts are unable to control pulmonary mycobacterial infection by M. bovis BCG bacilli as a result of the lack of both lymphocytic repertoire and NK cells (11). While these mice were found to have an impaired ability to form well-organized granuloma in their lungs, particularly at earlier stages of infection, the level of macrophage infiltration in the lung appeared normal (11). To determine whether there were any defects in macrophage functions, we compared both the number and function of alveolar macrophages between naive SCID beige and immune-competent control mice. As shown in Table IGo, SCID mice contained a similar number of macrophages compared with wild-type mice. Furthermore, these cells responded as well as those from control mice to GM-CSF stimulation in vitro in undergoing proliferation. Upon LPS stimulation or mycobacterial infection, SCID macrophages released similar amounts of TNF-{alpha}. These findings indicate that regardless of the lack of lymphocytes and NK cells, SCID beige mice have a normal macrophage population. This strain of mice thus provided us a unique tool to investigate the role of macrophage activation in host defense against mycobacterial infection in the absence of adaptive immune components.


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  		Table I. Characteristics of lung macrophages in naive SCID micea

 
Selectively impaired type 1 cytokine responses in the lung of SCID mice during pulmonary mycobacterial infection

To identify the cytokine that may be missing during antimycobacterial immune responses in SCID beige mice and thus could be chosen as a candidate for replacement to activate macrophages, we analyzed the level of type 1 cytokines IFN-{gamma}, IL-12, and TNF-{alpha} in the lung of SCID mice at day 37 postpulmonary mycobacterial infection. Compared with immune-competent mice, SCID mice had much less IFN-{gamma} and IL-12 in the lung, whereas the level of TNF-{alpha} was not diminished (Fig. 1Go). Since T cells, NK cells, and macrophages can all be a source of IFN-{gamma} (21, 22), diminished IFN-{gamma} responses most likely resulted from the lack of T and NK cells as well as the lack of macrophage activation. The unimpaired TNF-{alpha} response suggests that this cytokine was primarily released from macrophages, since we have recently shown that mycobacteria could directly release TNF-{alpha} from macrophages, independent of IL-12, in contrast to macrophage IFN-{gamma} release (19, 20), and that this cytokine by itself was unable to adequately activate macrophages to enhance their mycobactericidal activities. Thus, markedly weakened host defense against mycobacterial infection in SCID hosts is most likely attributable to inadequate responses of IFN-{gamma} and IL-12 and the subsequent lack of macrophage activation. Since IFN-{gamma} is a potent macrophage activator critically required for antimycobacterial host defense (20, 23, 24), we chose to reconstitute the level of this cytokine in the lung of SCID beige mice by using an intrapulmonary gene transfer approach.



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  		FIGURE 1. Type 1 cytokine profile in the lung of SCID beige mice following pulmonary mycobacterial infection. IFN-{gamma} (A), IL-12 (B), and TNF-{alpha} (C) were measured in BAL fluids collected from immune-competent control and SCID beige mice at day 37 postinfection. Results are expressed as mean ± SEM from four mice per strain. The difference in the levels of IFN-{gamma} or IL-12 is statistically significant (p < 0.01).

 
Repeatability of IFN-{gamma} gene transfer to the lung of SCID mice

Our previous studies have demonstrated that intrapulmonary adenoviral gene transfer leads to raised levels of cytokine product in the lung for a period of 10 to 12 days (16, 17, 18). Since pulmonary mycobacterial infection is a chronic process and we have demonstrated a sustained IFN-{gamma} response present in the lung of immune-competent hosts during pulmonary mycobacterial infection (11, 13), repeated IFN-{gamma} gene transfer to the lung may be required to sustain the level of IFN-{gamma} and to significantly enhance host defense in SCID mice. The lack of adaptive immune components in SCID mice would allow the efficient repeated adenoviral-mediated gene transfer. To establish the feasibility of such repeated gene transfer in SCID hosts, we delivered the first dose (4 x 108 PFU) of adenoviral IFN-{gamma} gene transfer vector, i.n. to naive SCID mice, and examined the level of IFN-{gamma} protein in the BAL fluid at days 4 and 14. Single gene transfer resulted in markedly increased levels of IFN-{gamma} by day 4 (2000 pg/ml), which declined to ~40 pg/ml by day 14 (Fig. 2GoA). However, if a second dose of IFN-{gamma} vector was given to such mice at day 10, the level of IFN-{gamma} rose back to ~2000 pg/ml again at day 14 (Fig. 2GoA). These findings thus indicate the feasibility of repeated IFN-{gamma} gene transfer for reconstituting and sustaining IFN-{gamma} expression in the lung of SCID mice, to a level similar to that in the lung of immune-competent mice during pulmonary mycobacterial infection (13).



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  		FIGURE 2. A, IFN-{gamma} level in the lung of naive SCID beige mice following repeated IFN-{gamma} gene transfer. A dose of AdIFN-{gamma} was delivered i.n. to mice, and the level of IFN-{gamma} in BAL fluid was examined at days 4 and 14 (post-1st i.n.). In other mice, a second dose of gene vector was given i.n. at day 10 after the initial i.n. gene transfer, and the level of IFN-{gamma} was examined at day 14 (post-2nd i.n.). B, The experimental regimen. SCID beige mice were infected intratracheally with live M. bovis BCG at day 0. At days 10 and 20 postinfection, mice were treated with a control vector (Addl) or IFN-{gamma} gene transfer vector (AdIFN-{gamma}). Groups of mice were sacrificed at days 27, 37, and 52, and samples were collected for analysis.

 
Enhanced host resistance to fatal pulmonary mycobacterial infection by postinfection i.n. IFN-{gamma} gene transfer to SCID mice

To examine whether IFN-{gamma} gene transfer modality could effectively activate macrophages in SCID beige mouse lung in a clinically relevant setting, pulmonary mycobacterial infection was established first in SCID mice, and then the initial dose of adenoviral IFN-{gamma} gene transfer vector was delivered i.n. to these mice 10 days after the establishment of infection. A second dose of IFN-{gamma} gene vector was given at day 20 postinfection to prolong the level of IFN-{gamma} (Fig. 2GoB, experimental regimen). Mice were sacrificed at days 27, 37, and 52 postinfection, and the level of infection was assessed by a colony enumeration assay.

SCID mice that were treated with a control vector failed to control infection and demonstrated uncontrollable, escalating replication of mycobacteria in the tissue (Fig. 3Go). In contrast, mice that were treated with IFN-{gamma} transgene demonstrated at least 10-fold lower bacterial counts at days 27 and 37, and 7.7-fold lower bacterial counts at day 52. Histologically, while loads of bacilli were easily located within macrophages in control mouse lung, much fewer bacilli were seen in the lung of mice treated with IFN-{gamma} transgene (Fig. 4Go, A and B). Of note, IFN-{gamma} transgene treatment only moderately increased the number of macrophages/monocytes in the lung (not shown) and did not markedly improve macrophage granuloma formation in the lung, in contrast to a vigorous granulomatous response seen in the lung of immune-competent mice (Fig. 4Go, C–E). These findings suggest that IFN-{gamma} transgene treatment improved host resistance primarily via its effect on the quality of macrophages in the lung of infected SCID mice.



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  		FIGURE 3. Level of mycobacterial infection in the tissue. Lungs were recovered from mice receiving the control (Addl) or IFN-{gamma} gene vector (AdIFN-{gamma}) at days 27, 37, and 52 postinfection, and subjected to a colony enumeration assay. Results are expressed as mean ± SEM from a total of five mice per group for day 27, nine mice per group for day 37, and four mice per group for day 52. The difference between the control and IFN-{gamma} transgene treatment is statistically significant for all time points (p = 0.0033 for day 27, p = 0.0005 for day 37, and p = 0.03 for day 52).

 


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  		FIGURE 4. Histopathology of SCID beige mouse lung. Lung tissues were recovered at day 37 postinfection from mice receiving the control Addl and IFN-{gamma} gene vector AdIFN-{gamma}. A, Addl-treated. Lung tissue sections were stained with Ziehl-Neelsen for intracellular mycobacteria (arrowheads). B, AdIFN-{gamma}-treated. Lung tissue sections were stained with Ziehl-Neelsen. C, Addl-treated. H&E staining. D, AdIFN-{gamma}-treated. H&E staining. E, Immune-competent C57BL/7 mouse lung. Note vigorous granuloma formation with heavy lymphocytic infiltration. H&E staining. b, Bronchial lumen in the lung. Magnification, x1890 (A and B) and x470 (C–E).

 
In separate experiments, we set out to evaluate whether enhanced immune protection by repeated IFN-{gamma} deliveries could prolong the survival of infected SCID mice. We have previously observed that untreated SCID mice will succumb to M. bovis BCG infection (11). Thus, we infected and treated SCID mice with the control vector or IFN-{gamma} gene vector (8–10 mice/group), as described in Fig. 2GoB, except that after the second dose of vector, additional three doses of vector were given, ~10 days apart, to prolong the level of IFN-{gamma} in the lung. The mortality of mice was monitored. We found that mice in control group became morbid around day 40 and started to die around day 50, and all mice died by day 80 with an average survival of approximately 60 days (Fig. 5Go). In comparison, mice treated with IFN-{gamma} transgene appeared healthy at earlier stages and did not start to die until ~70 days, and 25% of mice survived beyond 90 days with a significantly prolonged survival rate (Fig. 5Go).



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  		FIGURE 5. Prolonged survival of SCID beige mice by IFN-{gamma} gene transfer. Mice (8–10/group) were infected with M. bovis BCG and treated i.n. with the control vector (control) or AdIFN-{gamma} at days 10, 20, 30, 40, and 50, and monitored for their mortality.

 
Increased levels of IFN-{gamma} and IL-12, but not TNF-{alpha}, in the lung by IFN-{gamma} gene transfer

To investigate the mechanisms by which IFN-{gamma} transgene enhanced protection against mycobacterial infection in SCID beige mice, we examined the cytokine profile in the lung. The concentration of type 1 cytokines IFN-{gamma}, IL-12, and TNF-{alpha} in the BAL fluid was determined 7 days post-second IFN-{gamma} gene delivery (day 27 postinfection) (Fig. 2GoB). Following IFN-{gamma} gene transfer, a significant amount of IFN-{gamma} was measured in the lung, whereas it was barely detectable in the lung of control mice at this time point (Fig. 6GoA). The level of IL-12 was also elevated in the lung of IFN-{gamma} transgene-treated mice (Fig. 6GoB). Of interest, the level of TNF-{alpha} was similar in both control and IFN-{gamma} transgene-treated SCID mice (Fig. 6GoC), suggesting that IFN-{gamma} transgene expression selectively up-regulated the release of IL-12, but not TNF-{alpha}, in this model.



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  		FIGURE 6. Type 1 cytokine profile in the lung of Mycobacterium-infected SCID beige mice postintrapulmonary IFN-{gamma} gene transfer. IFN-{gamma} (A), IL-12 (B), and TNF-{alpha} (C) were measured in BAL fluids collected from SCID beige mice receiving the control vector (Addl) or IFN-{gamma} gene vector (AdIFN-{gamma}) at day 27 postinfection. Results are expressed as mean ± SEM from five mice per group. The difference in the level of IFN-{gamma} and IL-12 between two treatments is statistically significant (p = 0.00001 and p = 0.002, respectively).

 
Phenotypic activation of macrophages by IFN-{gamma} gene transfer in the lung

We next examined whether heightened type 1 cytokines enhanced antimycobacterial host defense by activating macrophages. To this end, we first examined the phenotype of lung macrophages isolated 7 days post-second gene transfer (27 days postinfection) from SCID mice for their surface expression of CD11b and MHC class I and II molecules. IFN-{gamma} is a potent inducer of MHC class I and II molecules. Macrophages express high density of CD11b and MHC class I and, when activated, MHC class II molecules (20). In this particular experiment, an adenoviral vector expressing murine GM-CSF was used as a control to compare with the vector expressing IFN-{gamma}. By a cytologic analysis conducted before FACS staining, we found that the majority of cells to be examined by FACS analysis were macrophages (95%, 80%, and 91% for adenoviral E1-deleted l70-3, adenoviral GM-CSF, and adenoviral IFN-{gamma}, respectively). Approximately 20% of cells from control vector-treated SCID mice expressed bright CD11b and MHC class I (Fig. 7Go, top panels). Expression of GM-CSF in the lung only slightly increased the size of this cell population (25%). In contrast, IFN-{gamma} gene transfer markedly increased this cell population expressing bright CD11b/MHC class I to 48%. A similar picture was seen with MHC class II staining (Fig. 7Go, bottom panels). While the number of macrophages expressing bright CD11b/MHC II was small and similar in both control and GM-CSF groups (1–2%), IFN-{gamma} transgene treatment increased this macrophage population to 9%. These findings suggest that compared with other macrophage stimuli such as GM-CSF, IFN-{gamma} is a uniquely potent macrophage activator during mycobacterial infection.



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  		FIGURE 7. Macrophage phenotype by FACS analysis. Cells were isolated and pooled from the lung of three to four SCID beige mice receiving the control vector (Addl70–3), vector expressing GM-CSF (AdGM-CSF), or vector expressing IFN-{gamma} (AdIFN-{gamma}) at day 27 postinfection. Cells were immunostained with labeled mAbs for CD11b, MHC class I, and MHC class II surface molecules, and analyzed on a FACScan.

 
Enhanced macrophage release of cytokines and NO by IFN-{gamma} gene transfer in the lung

To investigate whether lung macrophages phenotypically activated by IFN-{gamma} gene transfer released increased amounts of soluble mediators critical to antimycobacterial host defense, intraalveolar macrophages were isolated 7 days post-second i.n. gene transfer (day 27 postinfection) and cultured in vitro under various conditions. The release of both type 1 cytokines and NO was determined. We found that in contrast to those from control mice, macrophages from IFN-{gamma} transgene-treated mice spontaneously released large amounts of IFN-{gamma} (441 pg/ml vs 22 pg/ml in controls) and IL-12 (257 pg/ml vs 54 pg/ml in controls) (Fig. 8Go, A and B). Upon stimulation with LPS, these cells released further increased amounts of these cytokines. In comparison, macrophages from both control and IFN-{gamma} transgene-treated mice released comparable amounts of TNF-{alpha} spontaneously or upon LPS stimulation (Fig. 8GoC). These results suggest that cytokines measured in the lung were released primarily from activated macrophages since the profile of cytokine production in alveolar macrophages mirrors that in the lung (Fig. 6Go).



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  		FIGURE 8. Macrophage type 1 cytokine release ex vivo. Macrophages were isolated and pooled from the lung of several SCID beige mice receiving the control vector (Addl) or IFN-{gamma} gene vector (AdIFN-{gamma}) at day 27 postinfection. Cells were cultured in vitro for 3 days without stimulation (no) or with LPS. Supernatants were measured for IFN-{gamma} (A), IL-12 (B), and TNF-{alpha} (C). Results are expressed as mean ± SEM from three to five wells per condition of two independent experiments.

 
Since we and others have shown that IFN-{gamma} is a key inducer of NO in macrophages during mycobacterial infection and NO is a potent mycobactericidal chemical compound required for host defense (19, 25, 26), we further examined the ability of macrophages to release NO ex vivo. Macrophages from IFN-{gamma} transgene-treated mice spontaneously released a much greater amount of NO than those from controls (Fig. 9Go, A and B). Stimulation with LPS, IFN-{gamma}, or IL-12 further enhanced the release of NO. These findings suggest that IFN-{gamma} transgene treatment in SCID mice enhanced host defense against mycobacterial infection via its effect on macrophage activation, particularly NO release.



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  		FIGURE 9. Macrophage NO release ex vivo. Macrophages were isolated from the lung of SCID beige mice receiving the control vector (Addl control) or IFN-{gamma} gene vector (AdIFN-{gamma}) at day 27 postinfection. Cells were cultured in vitro for a period of 3 days without stimulation (no) or with LPS, IFN-{gamma}, or IL-12. The amount of NO was measured as nitrite by using a NO assay. Results are expressed as mean ± SEM from triplicates.

 
Enhanced mycobacterial phagocytosis and decreased mycobacterial replication in macrophages by IFN-{gamma} gene transfer in the lung

To examine whether activation of macrophages by IFN-{gamma} gene transfer to the lung translated to increased antimycobacterial activities in macrophages, macrophages were isolated from the lung of SCID beige mice that were treated i.n. with control vector or IFN-{gamma} gene transfer vector without mycobacterial infection. These cells were then allowed to interact with live mycobacteria in vitro, and their ability to phagocytose and to inhibit the growth of intracellular mycobacteria was analyzed. By using a phagocytosis assay, a greater number of macrophages from IFN-{gamma}-treated mice were found to have engulfed mycobacteria (Table IIGo). Furthermore, the average number of engulfed bacilli per macrophage was also greater in macrophages of IFN-{gamma} transgene group (3.41/macrophage vs 1.81/macrophage in control group).


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  		Table II. Antimycobacterial activities in IFN-{gamma}-activated lung macrophages of SCID mice

 
To examine whether following phagocytosis, activated macrophages could inhibit the growth of intracellular mycobacteria, a colony enumeration assay was conducted with macrophage cellular lysates collected at days 0 and 3 post in vitro infection. At day 0, the recovery of bacilli from IFN-{gamma}-activated macrophages was approximately 3 times greater than that from control macrophages (Table IIGo), again indicating that IFN-{gamma}-activated macrophages more avidly phagocytosed mycobacteria. By day 3, while the number of intracellular bacilli in control macrophages increased by ~141%, the number of bacilli in IFN-{gamma}-activated macrophages decreased by ~11% (Table IIGo). These findings indicate that IFN-{gamma} gene transfer-mediated macrophage activation endows macrophages with a greater ability to engulf mycobacteria and control their replication.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our current study has demonstrated: 1) SCID beige hosts have a relatively normal tissue macrophage population that provides a niche to allow the therapeutic manipulation of innate immunity; 2) during mycobacterial infection, the type 1 cytokine response is only selectively impaired in SCID mice with significantly weakened IFN-{gamma} and IL-12, but not TNF-{alpha}, responses; 3) repeated IFN-{gamma} gene transfer to SCID mouse lung, after the establishment of mycobacterial infection, enhanced host defense in the absence of lymphocytes and NK cells; and 4) IFN-{gamma} gene transfer enhanced host defense in SCID hosts, not through improving macrophage granuloma formation, but rather by activating macrophages. These observations demonstrate the rationale and feasibility to control the otherwise uncontrollable mycobacterial infection in immune-deficient humans by tailoring the innate immunity with macrophage-activating transgene-based therapeutics. To our knowledge, our study represents the first to demonstrate that IFN-{gamma} transgene-based therapeutic is effective in enhancing host defense against a fatal intracellular bacterial infection in the absence of T and B lymphocytes and NK cells.

Mycobacteria are facultative intracellular pathogens of macrophages. Increasing experimental evidence suggests that mycobacteria evade the intracellular bactericidal mechanisms of macrophages by interfering with the process of phagosome-lysosome fusion and the acidification of phagolysomal compartment (27). The ultimate control of mycobacterial replication and infection has been believed to involve several mechanisms of cell-mediated immunity (1, 2, 3, 4, 5, 6, 25, 26, 27, 28): 1) activation of infected macrophages by T and NK cell-derived cytokines, including IFN-{gamma} and TNF-{alpha}, and increased mycobactericidal activities in these activated macrophages; and 2) inhibition of mycobacterial replication and direct lysis of infected macrophages and mycobacteria by cytotoxic CD4 and CD8 T and NK cells via perforin/granzyme-, Fas/FasL-, and granulysin-mediated mechanisms. It has thus remained difficult to evaluate the relative contribution by macrophages themselves to the control of intracellular infection. Although RAG-1-/- and conventional SCID mouse strains are useful for addressing the role of T and B cells, the presence of NK cells in these mouse strains precludes a definitive conclusion about the contribution by macrophages. It has been found that these mice could provide certain protection, via NK cell-derived IFN-{gamma} and cytotoxic activities, against intracellular infection by listeria, Mycobacterium avium, Cryptosporidium parvum, and Shigella flexner (21, 29, 30, 31). By using a model of mycobacterial infection in SCID beige hosts deficient in both lymphocytes and NK cells, in conjunction with the use of an IFN-{gamma} gene transfer vector, we had a unique opportunity to investigate the role of macrophages, before and after activation, in host defense against mycobacterial infection.

We have previously found that compared with immune competent mice and mice with a selective defect in an immune component (IL-12, CD4, or CD8 T cells), SCID beige mice are the most susceptible to pulmonary mycobacterial infection by M. bovis BCG (11, 13, 14). These mice demonstrated diminished granuloma formation and progressively increased bacillary load in the tissue. In our current study, the level of IFN-{gamma} and IL-12 was found to be weakened, and in comparison, the level of TNF-{alpha} was unimpaired both in the lung and macrophages. Since these mice have a functionally normal macrophage population, as shown in our current study, the impaired granuloma formation cannot be attributed to any aberrant function of macrophages. Nor was it due to the lack of TNF-{alpha}, although this cytokine was shown to be required for granuloma formation during mycobacterial infection (32, 33, 34). The unimpaired TNF-{alpha} response to mycobacterial infection in the absence of T and NK cells suggests that first of all, this cytokine is macrophage derived, and second, this cytokine on its own is insufficient for granuloma formation and macrophage activation. Granuloma formation most likely requires both TNF-{alpha} and the presence of T cells. Indeed, macrophage TNF-{alpha} release in response to mycobacterial infection is independent of both IL-12 and IFN-{gamma} (19, 20). Markedly decreased IL-12 production concurrent with decreased IFN-{gamma} was somewhat unexpected in SCID beige mice, but is nonetheless in agreement with the notion that the optimal IL-12 release requires both IFN-{gamma} and intracellular pathogen (35, 36), a T cell-independent IL-12 release mechanism. T cell-dependent IL-12 release mechanism is apparently voided in SCID mice. The lack of IFN-{gamma} from both NK cells and T cells in SCID beige hosts accounts for the weakened IL-12 production. All of these findings indicate that when minimally activated in SCID beige hosts, macrophages are unable to control mycobacterial infection, and such inability is associated most closely with the lack of IFN-{gamma}, but not TNF-{alpha}.

Thus, to investigate whether macrophages in SCID beige hosts could be adequately activated to combat mycobacterial infection, an adenoviral gene transfer vector was chosen to express IFN-{gamma} in the lung. Such gene vector targets the transgene to airway epithelial cells and leads to a prolonged, raised level of transgene product in the lung of immune-competent mice (16, 17, 18). We have shown in the current study that the lack of adaptive immune components in SCID mice allows an even more sustained level of transgene product by allowing repeated gene transfer in the lung. Thus, we would be able to create an IFN-{gamma}-rich environment similar to that in an immune-competent host during mycobacterial infection (13). This strategy shall lead to the most efficient activation of macrophages. IFN-{gamma} transgene expression rendered after the establishment of infection led to heightened levels of IFN-{gamma} in the lung. As a result, the level of IL-12 was increased, reinforcing the notion that the optimal production of IL-12 requires IFN-{gamma} in a lymphocyte/NK cell-deficient host. Of importance, increased IFN-{gamma} was not accompanied by increased TNF-{alpha} nor by significantly improved granuloma formation, which again suggests that granuloma formation requires both type 1 cytokines and T cells. Airway expression of IFN-{gamma} transgene is remarkably effective in activating macrophages. Macrophages were activated to release increased amounts of not only IFN-{gamma}, but IL-12 as well as NO. They also expressed phenotypic activation markers, including MHC class I and class II molecules. These changes in macrophage activation status translated to increased antimycobacterial activities. These activated macrophage properties induced by IFN-{gamma} gene transfer in SCID mouse lung highly resemble those found in immune-competent mouse lung (20). The coupling of increased IFN-{gamma} release with increased IL-12 both in the lung and macrophages suggests that not only the optimal production of IL-12 requires IFN-{gamma} in SCID mice, but IL-12 may in turn release more IFN-{gamma} from macrophages since we have shown that macrophage IFN-{gamma} release requires two signals: mycobacteria and IL-12 (19, 20). Indeed, we found that activated macrophages spontaneously released increased amounts of both IFN-{gamma} and IL-12 ex vivo.

Hence, IFN-{gamma} transgene-mediated macrophage activation clearly led to enhanced control of mycobacterial replication in the absence of CD4 and CD8 T and NK cells, at least within the time frame of our current study. It is of importance to note that mycobacterial infection in SCID beige mice by attenuated M. bovis BCG is a very chronic process, different from that by virulent Mycobacterium tuberculosis, and the most effective control of infection may require a much more prolonged regimen with IFN-{gamma} transgene. In the mortality study presented in the current study, however, the IFN-{gamma} transgene regimen consisted of only five repeated doses starting from day 10 postinfection, 10 days apart thereafter, and a significant number of mice became moribund around day 80, a time when IFN-{gamma} transgene product from the last dosing tapered off. This very likely disallowed the persistence of enhanced immune protection and conferred only a limited prolonged survival in these IFN-{gamma} transgene-treated SCID mice. Thus, we expect that if the IFN-{gamma} transgene treatment continued beyond five repeated doses, these mice may experience a long-term survival. On one hand, our findings suggest that macrophages are a central effector cell in host defense against mycobacterial infection, and that the primary role played by T and NK cells in cell-mediated immunity is to warrant adequate macrophage activation by providing the key type 1 cytokine IFN-{gamma}. Recent studies with perforin or granzyme gene knockout mice seem to lend further support to this conclusion (37, 38). On the other hand, however, our findings also suggest that IFN-{gamma}-mediated macrophage activation alone, in the absence of T cells, may not be the entire answer to the eradication of intracellular mycobacteria (infection rebounds as IFN-{gamma} levels decline). The fact that prolonged IFN-{gamma} transgene expression in the lung of SCID beige mice, in the presence of endogenous TNF-{alpha}, fails to enhance granuloma formation suggests that T cells assist in granuloma formation, which, together with IFN-{gamma}, might be required for effective control of mycobacterial infection. This notion does not seem to support the conclusion from a recent study by Johnson and colleagues (39) that significant immunity to M. tuberculosis is not ablated in the absence of granuloma formation in ICAM-1-deficient mice.

Severely immune-compromised humans are susceptible to infection by normally otherwise nonpathogenic intracellular bacteria, particularly M. bovis BCG and environmentally borne mycobacterial strains. Although rIFN-{gamma} protein has been shown to provide certain therapeutic benefits to immune-competent hosts with M. tuberculosis or Mycobacterium leprae infection (40, 41), it will unlikely be effective in control mycobacterial infection in severely immune deficient hosts. Indeed, by systemic delivery of rIFN-{gamma} protein, Flynn and colleagues (23) could only minimally prolong the survival of IFN-{gamma}-deficient mice with M. tuberculosis infection. Our current study thus supports the local use of IFN-{gamma} transgene-based formulations to turn the innate immunity around in humans that are deficient in T cells or IL-12 or IL-12Rs (42, 43). Although still remained to be demonstrated, such IFN-{gamma} transgene therapeutic modality is expected to be even much more effective when used in conjunction with antimycobacterial chemotherapy.


   Acknowledgments
 
We thank Dr. Jay Kolls for the construction of AdIFN-{gamma} vector, Xueya Feng and Duncan Chong for amplification and purification of adenoviral vectors, and Michael Santosuosso for assistance in NO assay.


   Footnotes
 
1 This study is supported by operating grants from Canadian Institutes of Health Research and Ontario Thoracic Society, and by funds from McMaster University, St. Joseph’s Hospital, and Hamilton Health Sciences. J.W. is a fellow of from Canadian Institutes of Health Research/Canadian Lung Association. Z.X. holds from Canadian Institutes of Health Research New Investigator Award and Ontario Premier’s Research Excellence Award. Back

2 Address correspondence and reprint requests to Dr. Zhou Xing, Room 4H19, Department of Pathology and Molecular Medicine, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5 Canada. E-mail address: xingz{at}mcmaster.ca Back

3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; Ad, •••; Addl, •••; BAL, bronchoalveolar lavage; i.n., intranasal. Back

Received for publication February 28, 2001. Accepted for publication April 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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