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Published ahead of print on September 29, 2005, doi:10.1165/rcmb.2005-0198OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 167-173, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0198OC

CD4+ Cells Play a Limited Role in Murine Lung Infection with Mycobacterium kansasii

Catharina W. Wieland, Sandrine Florquin, Jennie M. Pater, Sebastiaan Weijer and Tom van der Poll

Laboratory of Experimental Internal Medicine, Department of Pathology, and Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine, and AIDS, Academic Medical Center, University of Amsterdam, The Netherlands

Correspondence and requests for reprints should be addressed to Catharina W. Wieland, Laboratory of Experimental Internal Medicine, G2-132, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: c.wieland{at}amc.uva.nl


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium that can cause severe infection in the immunocompromised host, especially in human immunodeficiency virus–infected patients. However, little is known about the pathogenesis of this infection. Because patients suffering from M. kansasii infection are severely compromised in their cellular immune response, we studied the course of infection in CD4+ cell knockout (KO) mice. Wild-type (WT) mice and CD4+ KO mice were infected with 105 cfu of M. kansasii. Although previously shown to be susceptible to Mycobacterium tuberculosis infection, CD4+ KO mice demonstrated no impairment in clearing infection with M. kansasii when compared with WT animals, despite reduced pulmonary inflammation (reduced granuloma formation and lymphocyte infiltration in the lungs). Pulmonary IFN-{gamma} levels and M. kansasii–induced IFN-{gamma} production by splenocytes from infected animals were reduced in CD4+ KO mice, confirming that these mice were defective in the M. kansasii–specific T helper cell type 1 immune response. Furthermore, mice deficient for IFN-{gamma}, IL-12p35, IL-12p40, or IL-18 also displayed a normal host defense against pulmonary infection with M. kansasii. These data suggest that CD4+ cells, IFN-{gamma}, and an intact T helper cell type 1 response play a limited role in protective immunity against pulmonary M. kansasii infection.

Key Words: CD4 • IFN-{gamma} • infection • mycobacterium • T helper cell type 1 response


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mycobacterium kansasii is one of the most frequent nontuberculous mycobacterial pathogens isolated from clinical specimens. Infection with M. kansasii can cause pulmonary disease similar to tuberculosis in patients with various immune deficiencies—in particular, in human immunodeficiency virus (HIV) infection or in patients with pre-existing pulmonary disease, like asthma and chronic obstructive pulmonary disease (1–4). Since the start of the acquired immunodeficiency syndrome (AIDS) epidemic, a vast increase in M. kansasii infection incidence has been observed (5). However, little is known about the pathogenicity, mode of transmission, and natural reservoir of M. kansasii. The organism has been recovered occasionally from rivers and lakes, but also from tap water, showerheads, and drinking water distribution systems, and is thought to be acquired from the environment rather than from contact with infected patients. Unlike Mycobacterium tuberculosis, culturing of M. kansasii from human sources is not exclusive proof of disease: as many as one-third of isolates has been reported to represent colonization or indolent infection of the respiratory tract rather than disease (6).

During pulmonary infection by M. tuberculosis or other pathogenic nontuberculous mycobacteria, like Mycobacterium avium complex, an appropriate T helper cell (Th) type 1 response is of utmost importance to restrain the infection (7, 8). Mice lacking CD4+ T cells, IFN-{gamma}, or IL-12p40 are highly susceptible to these mycobacterial infections (9–16). Because the Th1 response is severely impaired in patients with HIV infection, and because patients with AIDS are the main group suffering from M. kansasii infection, we were interested in determining whether the host response to M. kansasii is indeed dependent on a functional Th1 response. We therefore established a murine model of M. kansasii pulmonary infection, and subsequently compared the immune response in CD4+ knockout (KO) and normal wild-type (WT) mice. In contrast to pulmonary infection with M. tuberculosis or nontuberculous mycobacteria other than M. kansasii, the deficiency of CD4+ cells did not render the mice more susceptible to M. kansasii. Moreover, mice lacking Th1 mediators like IFN-{gamma}, IL-12p35, or IL-12p40 were not different from WT mice in their host defense response. These data lead us to hypothesize that M. kansasii infection can only develop in a complex form of immunodeficiency involving not only single mediators and/or cell types, but several different parts of the innate and adaptive immune system. For example, HIV-infected patients have been found to have reduced numbers and function of macrophages, dendritic cells, natural killer (NK) cells, NK T cells, and a diminished production capacity of proinflammatory mediators, such as chemokines and leukotrienes (17–22).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Mice
Six- to eight-week-old age- and sex-matched CD4+, IFN{gamma}, IL-12p35, and IL-12p40 KO mice on a C57BL/6 background and WT C57BL/6 control mice were purchased from Jackson Laboratory (Bar Harbor, ME). IL-18 KO mice, back-crossed six times to C57BL/6 background, were generated as described previously (23); age- and sex-matched C57BL/6 WT mice were used as their controls (Harlan Spague Dawley, The Hague, The Netherlands). The Animal Care and Use Committee of the University of Amsterdam approved all experiments.

Experimental Infection
M. kansasii, a clinical isolate obtained from a coal-mine worker presenting with tuberculosis-like disease, was grown for 2 wk in liquid Dubos medium containing 0.01% Tween-80. A replicate culture was incubated at 37°C, harvested at mid-log phase, and stored in aliquots at –70°C. For each experiment, a vial was thawed and washed with sterile 0.9% NaCl. Lung infection was induced as described previously (24–26). Briefly, mice were anesthetized by inhalation with isoflurane (Abott Laboratories Ltd., Kent, UK) and infected intranasally with M. kansasii in 50 µl saline. At the time points indicated in RESULTS, five to eight mice per group were killed, and lungs were removed aseptically and homogenized in 5 vols of sterile 0.9% NaCl. Ten-fold dilutions were plated on Middlebrook 7H11 agar plates to determine bacterial loads. Colonies were counted after 14–18 d at 37°C.

Cytokine and Chemokine Measurements
For cytokine measurements, lung homogenates were diluted 1:2 in lysis buffer containing 300 mM NaCl, 30 mM Tris, 2 mM MgCl2, 2 mM CaCl2, 1% Triton X-100, and pepstatin A, leupeptin, and aprotinin (all 20 ng/ml; pH 7.4), and incubated on ice for 30 min. Homogenates were centrifuged at 1,500 x g at 4°C for 15 min; supernatants were sterilized using a 0.22-µm filter (Corning Incorporated, Corning, NY) and stored at –20°C until assays were performed. IFN-{gamma}, IL-4, TNF, IL-1beta, cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein (MIP)-2 were measured using specific ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. For detection of IL-6, a Bio-Plex Cytokine Array was used (27) (Bio-Rad Laboratories, Inc., Hercules, CA). The detection limits were 62 pg/ml for IL-4, TNF, IL-1beta, and MIP-2, 32 pg/ml for IFN-{gamma}, 37 pg/ml for KC, and 7.8 pg/ml for IL-6.

Characterization of Inflammatory Infiltrates in the Lungs
Pulmonary cell suspensions were obtained by crushing lungs through a 40-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ) as described previously (24, 25). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4); the remaining cells were washed twice with RPMI 1640 (BioWhittaker, Verviers, Belgium), and counted using a hemocytometer (Fisher Emergo b.v., Amsterdam, The Netherlands). The percentages of macrophages, polymorphonuclear leukocytes, and lymphocytes were determined using cytospin preparations stained with hematoxylin and eosin.

Flow Cytometric Analysis
Lung cell suspensions obtained from infected mice were analyzed by flow cytometry using FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA), as described previously (24, 25). Cells were brought to a concentration of 4 x 106 cells/ml of FACS buffer (PBS supplemented with 0.5% BSA, 0.01% NaN3, and 0.35 mM EDTA). Immunostaining for cell surface molecules was performed for 30 min at 4°C using directly labeled antibodies against CD3 (CD3-phycoerythrin), CD4 (CD4-CyChrome), CD8 (CD8-FITC and CD8-peridinin chlorophyl protein), and CD69 (CD69-FITC). All antibodies were used in concentrations recommended by the manufacturer (Pharmingen, San Diego, CA). After staining, cells were fixed in 2% paraformaldehyde, and T cell surface molecules were analyzed on CD3+ cells within the lymphocyte gate.

Histology
Lungs for histology were harvested after infection, fixed in 10% buffered formaline, and embedded in paraffin. Four-micron sections were stained with hematoxylin and eosin, and analyzed by a pathologist who was blinded for groups. To score lung inflammation and damage, the entire lung surface was analyzed with respect to the following parameters: interstitial inflammation, endothelialitis, bronchitis, granuloma formation, and pleuritis. Each parameter was graded on a scale of 0–4 (0, absent; 1, mild; 2, moderate; 3, severe; and 4, very severe). The total "lung inflammation score" was expressed as the sum of the scores for each parameter, the maximum being 20.

Splenocyte Stimulation
Single-cell suspensions were obtained by crushing spleens through a 40-µm cell strainer (Becton Dickinson) as previously described (24, 25). Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4); the remaining cells were washed twice with RPMI 1640 (BioWhittaker) supplemented with 10% FCS and 1% antibiotic-antimycotic (GibcoBRL, Life Technologies, Rockville, MD). Cells were seeded in 96-well, round-bottom culture plates at a cell density of 1 x 106 cells/well in quadruplicate, and stimulated with 2 x 105 heat-killed (HK) M. kansasii (heat killing: 20 min in 80°C waterbath). As controls, splenocytes from uninfected WT mice and M. tuberculosis infected animals (intranasal inoculation with 105 CFU of M. tuberculosis H37Rv 5 wk earlier) were stimulated with 2 x 105 CFU of HK M. kansasii or purified protein derivative (PPD; Statens Seruminstitut, Copenhagen, Denmark), respectively. Supernatants were harvested after 48-h incubation at 37°C in 5% CO2, filter-sterilized, and stored at –20°C until ELISA was performed.

Statistical Analysis
All values are expressed as mean ± SEM. Comparisons were done with Mann-Whitney U tests using GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA). When comparing two groups at multiple time points, two-way ANOVA was used. Values of P < 0.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Host Defense against M. kansasii Infection Does Not Rely on CD4+ Cells
Host defense against mycobacterial infection, in particular tuberculosis, is strongly dependent on the presence of CD4+ T cells and IFN-{gamma}, the main type-1 cytokine produced by this cell type (7, 8). To determine the role of CD4+ T cells in the immune response to pulmonary infection with M. kansasii, we infected CD4+ KO and normal C57BL/6 WT mice with this organism. Before these experiments, we first established that C57BL/6 WT mice displayed progressively declining mycobacterial loads in their lungs during a 20-wk follow-up period after intranasal infection with M. kansasii at doses of up to 106 cfu; the infection rarely disseminated to distant organs: liver and spleen cultures were positive for mycobacteria only during the first weeks of infection, and bacterial counts were low (data not shown). For subsequent experiments with CD4+ KO mice, we chose an infectious dose of 105 cfu, anticipating that, whereas this dose would be effectively cleared by WT mice, this would not be the case in KO mice with a defective Th1 response. Much to our surprise, CD4+ KO mice demonstrated decreasing mycobacterial loads during an 8-wk follow-up period after intranasal infection with M. kansasii comparable to that observed in WT mice (Figure 1).


Figure 1
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  		Figure 1. CD4+ cells are not important for the clearance of Mycobacterium kansasii from the lungs. Mycobacterial loads in lungs: WT (closed symbols) and CD4+ KO (open symbols) mice were intranasally infected with 105 cfu of M. kansasii. After 1 d, 4 wk, and 8 wk of infection, mice were killed and bacterial loads were determined in lung homogenates. Data are means ± SEM of three (1 d) or eight mice per group at each time point.

 
CD4+ KO Mice Display Reduced Pulmonary IFN-{gamma} Levels
Like M. tuberculosis, M. kansasii is an intracellular pathogen residing in macrophages (28, 29). Therefore, pulmonary macrophages presumably play an important role in clearing this mycobacterium. IFN-{gamma} is a typical Th1 cytokine and considered a key cytokine in host defense against pulmonary M. tuberculosis (7, 8). Among other functions, IFN-{gamma} activates macrophages to phagocytose and kill intracellular pathogens (30, 31). We therefore measured IFN-{gamma} in lung homogenates of infected WT and CD4+ KO animals. At both 4 and 8 wk after infection, pulmonary IFN-{gamma} levels were reduced in CD4+ KO animals, pointing to a reduced local Th1 response (Figure 2). To gain more insight in the Th2 response in WT and CD4+ KO mice, we measured IL-4 in lung homogenates. At both time points, no differences in IL-4 levels were detected locally (Figure 2).


Figure 2
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  		Figure 2. CD4+ KO mice have reduced IFN-{gamma} lung levels. Pulmonary levels of IFN-{gamma} (A, B) and IL-4 (C, D) after 4 (A, C) and 8 (B, D) wk of infection with 105 cfu of M. kansasii. IFN-{gamma} and IL-4 were measured in lung homogenates. Data are means ± SEM of eight mice per group at each time point. *P < 0.05 versus WT mice.

 
Reduced Pulmonary Inflammation in CD4+ KO Mice
During M. tuberculosis infection, the formation of granulomas is of utmost importance to contain the infection. In mice, granulomas are formed by foamy macrophages and CD4+ and CD8+ lymphocytes (8). To obtain insight into the role of CD4+ cells in the generation of a pulmonary inflammatory response during M. kansasii infection, we compared cell recruitment and the cellular composition of lung infiltrates in CD4+ KO and WT mice using flow cytometry on whole–lung cell suspensions and histopathology. At 4 wk after infection, CD4+ KO mice displayed a reduced influx of lymphocytes to the site of infection and lower histopathologic scores (Tables 1 and 2). Whereas in WT mice granulomas were frequently present and well formed, CD4+ KO mice presented with a more diffuse infiltrate (Figure 3). Nevertheless, no differences between CD4+ KO and WT mice were detected with respect to whole-lung monocyte or neutrophil counts, or pulmonary cytokine and chemokine levels (Tables 2 and 3). As expected, no CD4+ lymphocytes were detectable in the lungs of CD4+ KO mice, whereas 66% of the lymphocytes in the lungs of WT animals stained positive for CD4 (Table 2). Despite the lack of CD4+ T cells, no difference in the number of CD8+ T cells recruited to the infected lungs was observed. The total number of lymphocytes expressing both CD8 and CD69, a marker of T-cell activation, did not differ between CD4+ KO and WT mice early after infection. At 8 wk after infection, no differences in histopathologic scores were detected (Table 1). At this time point, histopathology revealed inflammation of the lungs of the CD4+ KO mice comparable to that of the WT animals. The infiltrate of all mice consisted primarily of mononuclear cells (monocytes and lymphocytes), which primarily surrounded vessels and bronchi, and granulomas were less well demarcated (Figure 3). At 8 wk after infection, pulmonary levels of TNF, IL-1beta, KC, and MIP-2 were reduced in CD4+ KO animals when compared with WT animals (Table 3).


Figure 3
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  		Figure 3. Reduced granuloma formation and lymphocyte influx in lungs of CD4+ KO mice early in infection. Representative slides of lungs of WT (A, C) and CD4+ KO mice (B, D) infected with 105 cfu of M. kansasii 4 (A, B) and 8 (C, D) wk earlier. After 4 wk of infection, lungs of CD4+ KO mice showed reduced inflammation and granuloma formation. At 8 wk after infection, no difference in histopathology was observed. Hematoxylin and eosin staining; original magnification: x10.

 

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  		TABLE 1. INFLAMMATION SCORES

 

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  		TABLE 2. TOTAL AND DIFFERENTIAL CELL COUNTS

 

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  		TABLE 3. CYTOKINE AND CHEMOKINE PROFILE DURING Mycobacterium kansasii INFECTION

 
The Immune Response to M. kansasii Infection Is Not Dependent on IFN{gamma}, IL-12, or IL-18
The production of IFN-{gamma} is stimulated by IL-12, particularly in the presence of IL-18 (32). In line with the important role for IFN-{gamma} in host defense against M. tuberculosis, not only IFN-{gamma} KO mice, but also p35 KO, p40 KO, and IL-18 KO mice all demonstrated a reduced resistance against infection with this mycobacterium (11–13, 33–35). To obtain further proof of our finding that the Th1 response, and thus IFN-{gamma}, do not contribute to host defense against M. kansasii infection, we infected IFN-{gamma} KO, p35 KO, p40 KO, and IL-18 KO mice with 105 cfu of this microorganism and determined mycobacterial loads in their lungs 4 wk thereafter. No differences were found between the number of M. kansasii CFU in lungs of these KO mice and lungs of WT mice (Figure 4). In a second experiment, we infected WT and IFN-{gamma} KO mice with 105 CFU of M. kansasii and followed them for 12 wk. No differences in bacterial loads were detected between WT and IFN-{gamma} KO mice during this observation period (Figure 5). Of note, the presence of p35, p40, and IL-18 was necessary for an intact antigen-specific IFN-{gamma} production by infected splenocytes (i.e., splenocytes harvested from p35 KO, p40 KO, and IL-18 KO mice 4 wk after infection released significantly less IFN-{gamma} upon stimulation with heat-killed M. kansasii than splenocytes obtained from WT mice [Figure 6]). This difference in IFN-{gamma} production was comparable to the reduced production of IFN-{gamma} in CD4+ KO splenocytes upon stimulation with heat-killed M. kansasii (Figure 6A). Compared with PPD-induced IFN-{gamma} production by PPD-stimulated splenocytes obtained from M. tuberculosis–infected WT mice, IFN-{gamma} levels were low in culture supernatants of HK M. kansasii–stimulated splenocytes from M. kansasii–infected animals (Figure 6F). Nevertheless, in all cases, the amount IFN-{gamma} produced by splenocytes from infected WT animals was significantly increased compared with the IFN-{gamma} produced by spleen cells from uninfected mice (Figure 6F). To study the local IFN-{gamma} response, we also measured IFN-{gamma} in lung homogenates obtained from infected KO and WT mice. In contrast to IFN-{gamma} production by splenocytes, pulmonary IFN-{gamma} concentrations did not differ between WT and p35, p40, and IL-18 KO mice, whereas, as expected, no IFN-{gamma} was detected in lungs of IFN-{gamma} KO animals (data not shown). Histopathologically, the composition of the cellular infiltrates (determined by FACS) and pulmonary cytokine and chemokine levels did not differ between WT and IFN-{gamma} KO, p35 KO, p40 KO, and IL-18 KO mice (data not shown).


Figure 4
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  		Figure 4. Deficiency of IFN-{gamma}, IL-12p40, IL-12p35, or IL-18 does not influence mycobacterial load in lungs. Mycobacterial loads in lungs: WT (closed bars) and IFN-{gamma} KO (A), IL-12p35 KO (B), IL-12p40 KO (C), and IL-18 KO (D) (all KO, open bars) were infected intranasally with 105 Mycobacterium kansasii CFU. After 4 wk of infection, mice were killed and bacterial loads were determined in lung homogenates. Data are means ± SEM of six to eight mice per group.

 

Figure 5
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  		Figure 5. IFN-{gamma} is not important for the long-term clearance of M. kansasii from the lungs. Mycobacterial loads in lungs: WT (closed symbols) and IFN-{gamma} KO (open symbols) were intranasally infected with 105 cfu of M. kansasii. After 1 d, and 1, 2, 4, 6, 9, and 12 wk of infection, mice were killed and bacterial loads were determined in lung homogenates. Data are means ± SEM of three (1 d) or five mice per group at each time point. For comparison of groups, two-way ANOVA on log transformed data was used, and bacterial loads were not found to differ significantly.

 

Figure 6
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  		Figure 6. Reduced IFN-{gamma} response to M. kansasii–specific antigens by splenocytes. Splenocytes were isolated from infected WT (closed bars) and CD4+ KO (A), IFN{gamma} KO (B), IL-12p35 KO (C), IL-12p40 KO (D), and IL-18 KO (E) mice, and stimulated with heat-killed (HK) M. kansasii for 48 h. Control stimulations: stimulation with HK M. kansasii of splenocytes from naive mice (F, right bar) and stimulation with purified protein derivative (PPD) of splenocytes from WT mice intranasally infected with M. tuberculosis 5 wk earlier (F, left bar). IFN-{gamma} was measured in supernatants and expressed in pg/ml. Data are mean ± SEM of four to eight mice per group. ND, not detectable. **P < 0.01 and ***P < 0.001 versus WT.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Although M. kansasii is an emerging pathogen in immunocompromised patients, little is known about host defense mechanisms against this organism. Previous models of murine M. kansasii infection include the susceptible beige mouse strain or thymectomized C57BL/6 mice and applied intravenous or subcutaneous injection as the route of infection (36–41). Here we established a model of pulmonary infection with M. kansasii. After intranasal inoculation, the bacterium was able to persist in the lung of immune-competent WT C57BL/6 mice during a 20-wk follow-up period, with no evidence of a proliferative phase; the infection did not cause severe disease resembling the colonization observed in immune-competent humans. For comparison, immune-competent WT C57BL/6 mice infected with the nonpathogenic mycobacterial strain Mycobacterium smegmatis clear the infection during the first 10 d of infection (26). Moreover, this particular strain of M. kansasii was able to induce pulmonary disease similar to tuberculosis in healthy coal mine workers, indicating that this strain is able to cause disease, at least in humans.

Immunocompromised and, in particular, HIV-infected patients are susceptible to developing clinical disease after infection with M. kansasii (1, 3, 5, 42, 43). To mimic the immune-deficient state of HIV-infected patients, we used mice homozygous for the targeted mutation of CD4, that have a significant block in CD4+ T cell development: 90% of their circulating T cells are CD8+ (44). The CD4+ KO strain has been used as a model for advanced HIV infection and severe immunocompromised states before, and is a helpful tool when long-term absence of CD4+ T cells is studied (45, 46). Although CD4+ T cells are considered to be critical helper cells in inducing adaptive immune responses against mycobacteria other than M. kansasii (9, 15, 16, 47), only minor differences between normal WT animals and CD4+ KO mice were detected during 8 wk of pulmonary infection with M. kansasii. Our data are in contrast with those of a study performed by Flory and colleagues, in which thymectomized C57BL/6 mice treated with an anti-CD4 antibody displayed higher bacterial loads in lungs and spleens after intravenous infection with M. kansasii, persisting throughout a 12-wk observation period (40). Possible explanations for this discrepancy with our data are the route of infection (intravenous versus intranasal) and differences in virulence of the M. kansasii strains used.

At 4 wk after infection, 75% of all lymphocytes present in the lungs of CD4+ KO mice were CD8+ T cells. We presume that the remaining 25% of CD3+ cells were {gamma}{delta}T cells or major histocompatability complex class II restricted CD8–/CD4– {alpha}beta+ T helper cells (9, 48). It has been shown that {gamma}{delta}T cells undergo expansion in response to mycobacterial antigens in vitro and in vivo (49, 50), and the number of {gamma}{delta}T cells was increased during M. bovis BCG infection in beta2-microglobulin gene–deficient mice that lack functional CD8+ T cells (51). {gamma}{delta}T cells, as well as major histocompatability complex class II restricted CD8–/CD4– {alpha}beta+ T helper cells, CD8+ T cells, NK cells, and macrophages, are capable of producing IFN-{gamma}, and this IFN-{gamma} possibly compensated for the lack of CD4+ T cell help, allowing control of M. kansasii infection. To investigate the role of all CD3+ T and B cells in host defense against M. kansasii, the use of recombination activating gene KO mice is a possibility. In this study, we chose to investigate only CD4 T cell–deficient mice, as this mouse strain more closely mimics the immunodeficiency of HIV infection, a major risk factor for developing M. kansasii infection.

IFN-{gamma} has been implicated as a pivotal mediator of host defense against intracellular pathogens (52). The main cytokine that induces the proliferation and differentiation of T cells toward IFN-{gamma}–producing Th1 cells is IL-12, a heterodimeric cytokine that consists of a p35 subunit and a p40 subunit (IL-12p70) (53). IL-18 synergizes with IL-12p70 in promoting IFN-{gamma} induction in T cells and NK cells (32). Sufficient production of IFN{gamma} is crucial in host defense against M. tuberculosis and, in line with the IFN-{gamma}–inducing properties of IL-12 and IL-18, not only IFN-{gamma} KO mice, but also p35 KO, p40 KO, and IL-18 KO mice demonstrated a reduced resistance against infection with this mycobacterium (11–13, 33–35, 54). In addition, investigations that studied the role of IFN-{gamma} and IL-12 in host defense against M. avium found a similarly reduced resistance in mice deficient for either one of these cytokines (14, 55, 56). Furthermore, infection with M. bovis BCG was detrimental for mice deficient for IFN-{gamma}, IL-18, or p35/p40, resulting in higher bacterial burdens, increased inflammation in the lungs, and a reduced Th1 response (34, 54; reviewed for IFN-{gamma} KOs in Ref. 57). These results contrast with our current findings. Indeed, much to our surprise, even the total absence of IFN-{gamma} did not lead to increased susceptibility to pulmonary M. kansasii infection. After M. kansasii infection of p35, p40, and IL-18 KO mice, a similar picture as that observed in CD4+ KO mice was obtained: decreased IFN-{gamma} production after ex vivo stimulation of splenocytes and, thus, a reduced Th1 response to M. kansasii, but no differences in bacterial clearance. IFN{gamma} levels produced by splenocytes obtained from infected WT mice were low compared with the concentrations produced by splenocytes obtained from M. tuberculosis–infected animals. Nevertheless, the amount IFN-{gamma} produced by infected WT animals was significantly higher than IFN-{gamma} produced by splenocytes from uninfected WT mice. Of note, in contrast to the reduced pulmonary IFN-{gamma} concentrations in CD4+ KO mice, lung IFN{gamma} levels were unaltered in p35, p40, and IL-18 KO mice, suggesting that the local production of IFN-{gamma} during M. kansasii infection does not rely on IL-12 or IL-18. Altogether, these data indicate that host defense against M. kansasii is regulated by mechanisms that, at least in part, differ from the IFN-{gamma}–dependent mechanisms that are indispensable for protective immunity against other mycobacteria. Possibly, this striking difference is related to the relatively low virulence of M. kansasii in the immune-competent host.

Our data suggest that an intact CD4+ T cell– and IFN-{gamma}–driven immune response is not essential for host defense against M. kansasii in mice. By which mechanisms, then, is M. kansasii cleared from the lungs? It is conceivable that innate immune responses that are mediated by Toll-like receptors (TLRs), complement and antibodies, can have evoked a response that is strong enough to slowly clear the pathogen from the lung. Activated resident cells, such as alveolar and interstitial macrophages, as well as epithelial cells, produce cytokines and chemokines that attract and activate other inflammatory cells, like macrophages, monocytes, neutrophils, and T cells. Possibly, these cells were sufficiently activated to phagocytose and kill M. kansasii bacilli in the absence of a strong local and systemic IFN-{gamma}–driven adaptive immune response. This explains why the different strains of KO mice used in this study were not more vulnerable to M. kansasii infection, but were susceptible to M. tuberculosis infection. In this respect, it is interesting to note that we recently demonstrated that TLR-2 is important for the effective clearance of the nonpathogenic M. smegmatis from mouse lungs (26). Furthermore, we recently found that absence of IL-1 activity in IL-1 receptor type 1 KO mice lead to significantly reduced clearance of M. kansasii from the mouse lung, accompanied by an enhanced pulmonary inflammatory response (C.W.W., unpublished data). These novel findings further strengthen the notion that local innate host response and innate clearance mechanisms are sufficient to eliminate pulmonary infection with this virulent strain of M. kansasii.

In this study, we used CD4+ T cell KO mice as a model for HIV infection. Although CD4 depletion is a major characteristic of HIV pathogenesis, not only the adaptive immune response is disturbed. In HIV-infected patients, NK cells and NK T cells are depleted, and the number and function of circulating dendritic cells have been inversely correlated to viremia (18). Moreover, peripheral blood neutrophils, monocytes, and alveolar macrophages from patients with HIV infection have been reported to be defective in their capacity to produce leukotrienes, which are important proinflammatory mediators in the lung (17). Co-infection with non-HIV pathogens and associated TLR triggering have been postulated to be important exogenous factors that influence the severity and rate of disease progression in HIV+ individuals (58).

Our study reveals that the protective immune response against pulmonary infection with M. kansasii is not dependent on the presence of CD4+ T cells or production of IFN-{gamma}. These data lead us to hypothesize that M. kansasii infection can only develop in a complex form of immunodeficiency that cannot be copied by using KO mice lacking specific parts of the cellular immune system.


   Acknowledgments
 
The authors thank Ingvild Kop and Joost Daalhuisen for expert technical assistance.


   Footnotes
 
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0198OC on September 29, 2005

Received in original form May 26, 2005

Accepted in final form August 25, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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