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The Journal of Immunology, 2005, 174: 1027-1036.
Copyright © 2005 by The American Association of Immunologists

Distinct Roles for IL-4 and IL-10 in Regulating T2 Immunity during Allergic Bronchopulmonary Mycosis1

Yadira Hernandez*,{dagger}, Shikha Arora*, John R. Erb-Downward*,{dagger}, Roderick A. McDonald*, Galen B. Toews* and Gary B. Huffnagle2,*,{dagger}

* Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and {dagger} Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-{gamma} levels remained the same. TNF-{alpha} and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The fungal pathogen Cryptococcus neoformans enters the body through inhalation and establishes a primary pulmonary infection. Inbred mouse models of pulmonary cryptococcosis indicate a genetic component to the response. CBA/J and C.B-17 mice clear the infection faster than BALB/c mice. C57BL/6 mice can develop a chronic pulmonary infection (1, 2, 3, 4). Clearance of C. neoformans in resistant hosts involves the development of T1 cell-mediated immunity (5, 6, 7, 8, 9). T cells recruit and activate phagocytic effector cells, such as macrophages, against C. neoformans inducing a granulomatous reaction and the development of delayed-type hypersensitivity responses (10). Many cytokines, mainly T1 cytokines, have been shown to play important roles in clearance of C. neoformans. Among these cytokines are IFN-{gamma}, IL-2, IL-12, IL-18, TNF-{alpha}, and IL-15, and the CC chemokine ligands type 2 and type 3 (3, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22).

Chronic fungal infections can develop when the T1/T2 balance of cellular immunity is shifted away from T1 toward T2 immune responses (23). C57BL/6 mice, 6- to 8-wk-old at the time of infection, develop a nonresolving pulmonary fungal infection, which involves the up-regulation of T2 immunity. C. neoformans chronic infection stays primarily localized in the lungs of C57BL/6 mice, with minimal dissemination to extrapulmonary sites such as the spleen and the CNS. When compared with CBA/J or C.B-17 mice, susceptibility of C57BL/6 mice does not correlate with the level of inflammation at the site of infection, but does correlate with high levels of IL-5 secretion, low levels of IFN-{gamma}, and low levels of IL-2 production (2, 3, 4). Elevated levels of IL-5 in the lungs of C57BL/6 mice promote the development of pulmonary eosinophilia, which results in eosinophil-mediated tissue damage in the lungs, including deposition of eosinophilic YM1 crystals (4, 24). The level of susceptibility in C. neoformans infection correlates with the number of eosinophils infiltrating the lungs (4). Susceptible C57BL/6 mice have a large number of eosinophils in their lungs, moderately resistant BALB/c mice have transient influx of eosinophils, and highly resistant CBA/J mice have only a few eosinophils in their lungs (4). Thus, low dose infection of 6- to 8-wk-old C57BL/6 mice with C. neoformans strain 24067 produces a chronic allergic bronchopulmonary mycosis (ABPM).3 This model has been used to address the role of immunomodulatory agents such as OX40, Mycobacterium bacillus Calmette-Guérin, {alpha}-galactosylceramide (a CD1 ligand), IL-5 antagonists and anti-capsular Abs in addition to antifungal drugs in modulating immunity and promoting protective host responses (25, 26, 27, 28, 29, 30, 31, 32). Because both IL-4 and IL-10 can play significant regulatory roles in T2 responses to purified allergens (33, 34), our objective was to investigate the role of IL-4 and IL-10 in the development and manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes (LALN) in this model of ABPM.


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

Female IL-4 knockout (KO), IL-10 KO, and wild-type (WT) mice on a C57BL/6 background (16 ± 2 g) were obtained from The Jackson Laboratory. Mice were 6- to 8-wk-old at the time of infection. Mice were housed in sterilized cages covered with a filter top. Food and water were given ad libitum. The mice were maintained by the Unit for Laboratory Animal Medicine, University of Michigan, in accordance with regulations approved by the University of Michigan Committee on the Use and Care of Animals.

C. neoformans culture

C. neoformans strain 24067 (52D) was obtained from the American Type Culture Collection. For injection, yeast were grown to stationary phase (48–72 h) at 37°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco) on a shaker. The cultures were then washed in nonpyrogenic saline, counted on a hemocytometer, and diluted to 3.3 x 105 CFU/ml in sterile nonpyrogenic saline.

Surgical intratracheal inoculation

Mice were anesthetized by i.p. injection of pentobarbital (0.074 mg/g weight of mouse) and restrained on a small surgical board. A small incision was made through the skin over the trachea and the underlying tissue was separated. A 30-gauge needle was bent and attached to a tuberculin syringe filled with diluted C. neoformans culture. The needle was inserted into the trachea, and 30 µl of inoculum (104 CFU) were dispensed into the lungs. The needle was removed and the skin closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma.

CFU assay

For lung and LALN CFU, small aliquots were collected from lung digests or lymph node suspensions, respectively (described below). Aliquots of the lungs and lymph nodes were plated out on Sabouraud dextrose agar plates in duplicate 10-fold dilutions and incubated at room temperature. C. neoformans colonies were counted 2–3 days later, and the number of CFU per organ was calculated.

Induction of T cell deficiency

Mice were treated with 300 µg of anti-CD4 plus 300 µg of anti-CD8 mAb (GK1.5 and YTS 169.4, respectively) or anti-CD4 alone or anti-CD8 alone on day 0 of the infection and boosted with 100 µg of each mAb at days 7 and 14. T cell depletion was analyzed by flow cytometry of spleen cells. Depletion was >99% for CD4+ T cells and >95% for CD8+ T cells (data not shown).

Lung leukocyte isolation

Individual lungs were excised, minced, and enzymatically digested for 30 min in 15 ml of digestion buffer (RPMI 1640, 5% FCS, antibiotics, 1 mg/ml collagenase, and 30 µg/ml DNase). The cell suspension and undigested fragments were further dispersed by drawing up and down 20 times through the bore of a 10-ml syringe. The total cell suspension was then pelleted, and the erythrocytes were lysed by resuspending them in ice-cold NH4Cl buffer (0.83% NH4Cl, 0.1% KHCO3, and 0.037% Na2 EDTA, pH 7.4). Ten-fold excess of medium was added to return the solution to isotonicity. The isolated leukocytes were repelleted and resuspended in complete medium. Total lung leukocyte numbers were assessed in the presence of trypan blue using a hemocytometer.

Lung leukocyte subsets

Macrophages, neutrophils, and eosinophils were visually counted in Wright-Giemsa-stained samples of lung cell suspensions cytospun onto glass slides (Thermo Shandon Cytospin). For Wright-Giemsa staining, the slides were fixed for 2 min with a one-step methanol-based Wright-Giemsa stain (Harleco; EM Diagnostics Systems) followed by steps two and three of the Diff-Quik whole blood stain kit (Diff-Quik; Baxter Scientific Products). A total of 200–300 cells were counted from randomly chosen high power microscope fields for each sample. The percentage of a leukocyte subset was multiplied by the total number of leukocytes to give the absolute number of that type of leukocyte in the sample.

Numbers of B, CD4, and CD8 T cells were determined by flow cytometry. Lung leukocytes (5 x 105) were incubated for 30 min on ice in a total volume of 120 µl of staining buffer (FA buffer; Difco), 0.1% NaN3, and 1% FCS. Each sample was incubated with 1 µg of the respective FITC- or PE-labeled mAb (BD Pharmingen), or isotype-matched rat IgG. The samples were washed in staining buffer and fixed in 1% paraformaldehyde (Sigma-Aldrich) in buffered saline. Stained samples were stored in the dark at 4°C until analyzed on a flow cytometer (Coulter). The percentage of a lymphocyte subset was multiplied by the total number of leukocytes to give the absolute number of that type of lymphocyte in the sample.

Histology

Lungs were fixed by inflation with 10% neutral-buffered formalin. After paraffin embedding, 5-µm sections were cut and stained with H&E, periodic acid-Schiff, to stain mucus and mucus-secreting goblet cells, or Masson’s trichrome (collagen deposition stains blue).

Preparation of lymph nodes

LALN from two to three mice were pooled and processed into a cell suspension by gently passing tissues through nylon mesh. Cells were then washed and resuspended in complete RPMI 1640 medium. Total viable cell numbers were assessed by trypan blue exclusion and counted on a hemocytometer.

Lung leukocyte and lymph node cultures

Isolated lung leukocytes or lymph node cells (5 x 106/ml) were cultured for 24 h in 24-well plates with 2 ml of complete RPMI 1640 medium at 37°C and 5% CO2 with or without additional stimulus. Cultures were supplied with heat-killed C. neoformans (HKC) at 1 x 107/ml where indicated. Positive controls were performed by incubating the cells in the presence of 5 µg/ml Con A.

Cytokine production

Culture supernatants were harvested at 24 h and assayed for IFN-{gamma}, IL-4, IL-5, IL-13, TNF-{alpha}, IL-12, and IL-10 production by sandwich ELISA using the manufacturer’s instructions supplied with the cytokine-specific kits (BD Pharmingen and R&D Systems).

Total serum IgE

Serum was obtained by tail vein bleed of the mice and spinning the blood to obtain the serum. Serum samples were then assayed using an IgE-specific sandwich ELISA (BD Pharmingen).

Statistics

Statistical significance was calculated using ANOVA test (least significant difference posthoc) with significance being p < 0.05 for comparison between WT and IL-4 KO or WT and IL-10 KO. All values are reported as mean ± SEM for each group of pooled data derived from two to four experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pulmonary growth and dissemination of C. neoformans to the LALN

The first objective was to determine whether IL-10 and/or IL-4 play a role in the development of a chronic pulmonary C. neoformans infection. C57BL/6 WT, IL-4 KO, and IL-10 KO mice were inoculated intratracheally with 104 CFU of C. neoformans. The pulmonary burden of C. neoformans increased over 100-fold in all three groups of mice during the first week of infection. In WT mice, the number of cryptococci in the lungs remained elevated (> log 106 CFU) through weeks 2–3 (Fig. 1A). This is consistent with previously published studies demonstrating that C57BL/6 mice are unable to clear a pulmonary C. neoformans infection (3, 4). In contrast, IL-4 KO and IL-10 KO mice began to clear the infection during weeks 2–3. By week 3, the number of cryptococci in the lungs of IL-4 KO and IL-10 KO mice was 500- to 1000-fold lower than in WT mice (Fig. 1). Thus, in contrast to the chronic infection in WT mice, pulmonary clearance of C. neoformans was significantly greater in IL-4 KO and IL-10 KO mice, suggesting that production of IL-4 and IL-10 can promote the development of the chronic pulmonary infection. These data also demonstrate that effector mechanisms are not inherently defective in C57BL/6 mice because IL-4 KO and IL-10 KO C57BL/6 mice are able to clear the infection.



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  		FIGURE 1. Pulmonary growth of C. neoformans and dissemination to the lung-associated lymph nodes. A, Lung CFU were measured following C. neoformans intratracheal inoculation (104 CFU). Dotted line represents initial inoculum. B, LALN were removed, processed and plated for CFU. *, p < 0.05, comparing WT vs IL-10 KO or IL-4 KO mice; n = 11–12 mice/group/time point for weeks 1 and 2 and 6 mice/group/time point for week 3.

 
LALN CFU were measured to determine whether IL-4 and/or IL-10 facilitate the early dissemination of C. neoformans from the lungs to the draining lymph nodes. Under microisolator specific pathogen-free housing, LALN are undetectable (<104 lymph node cells) in uninfected mice. LALN expand following infection and are detectable by day 5–7 of infection. At this earliest time point, cryptococci were already detectable in the LALN of all three groups of mice. There was no significant difference in LALN CFU among WT, IL-4 KO, and IL-10 KO mice at any time point (Fig. 1B). LALN CFU did increase slightly between weeks 1 and 2 and then decreased between weeks 2 and 3 but was consistently within the range from 2.9 to 4.0 log CFU during the first 3 wk of infection. Thus, dissemination of C. neoformans from the lungs to the draining lymph nodes occurs early and is independent of the production of IL-4 and IL-10 by the host. Furthermore, these data also indicate that viable organisms can be found in the lymph nodes at the time that T cell expansion and priming are occurring.

Effect of T cell depletion in clearance of C. neoformans in IL-4 KO and IL-10 KO mice

IL-4 KO and IL-10 KO mice were depleted of CD4 and CD8 T cells to determine whether the enhanced clearance of C. neoformans in IL-4 KO and IL-10 KO mice was due to effects on the T cell-mediated response and not due to direct effects on potential innate clearance mechanisms. Pulmonary clearance of C. neoformans and IL-4 KO and IL-10 KO mice did not occur until weeks 2–3 postinfection (Fig. 1A), consistent with the need for development of a T cell-mediated immune response. Mice were rendered CD4 and CD8 T cell deficient by treatment with anti-CD4 and anti-CD8 mAbs every 7–8 days beginning at the onset of infection. CD4 and CD8 T cell depletion was >95% in these animals (data not shown). T cell replete WT mice were unable to clear C. neoformans from their lungs (Fig. 2) and depletion of CD4 and CD8 T cells did not significantly alter the pulmonary cryptococcal burden in WT mice (Fig. 2). In contrast, depletion of both CD4 and CD8 T cells abrogated pulmonary clearance in both IL-4 KO and IL-10 KO mice (Fig. 2). Pulmonary CFU in CD4/CD8 T cell-depleted KO mice was identical with that in WT mice (Fig. 2). Depletion of either CD4 or CD8 T cells alone in both IL-4 KO and IL-10 KO mice also significantly diminished clearance (Fig. 2). These data demonstrated that both CD4 and CD8 T cells were required for clearance during the protective response generated in IL-4 KO and IL-10 KO C57BL/6 mice, similar to the requirement of both T cell subsets during protective immunity in other inbred mouse strains (10). These data indicate that clearance in IL-4 KO and IL-10 KO mice requires T cells and the effect of IL-4 or IL-10 deficiency is not simply a direct up-regulation of a non-T cell effector mechanism.



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  		FIGURE 2. Effect of T cell depletion on C. neoformans pulmonary growth. WT and KO mice were depleted in vivo of CD4, CD8, or both CD4 and CD8 T cells using mAbs. Lungs were harvested at week 3 and aliquots from lung digests were plated for CFU. *, p < 0.05 comparing nondepleted to depleted mice within the same genetic background of mice (WT, IL-4 KO, or IL-10 KO); n = 3–8 mice/group.

 
Analysis of the pulmonary inflammatory response in IL-4 KO and IL-10 KO mice

To determine the mechanisms underlying IL-4 and IL-10 regulation of adaptive T cell immunity, the pulmonary inflammatory response was analyzed in IL-4 KO and IL-10 KO mice following infection with C. neoformans. Lung leukocytes were isolated by enzymatic digestion. Total lung leukocytes were enumerated at weeks 1, 2, and 3 after infection. WT, IL-4 KO, and IL-10 KO mice all developed inflammatory responses by week 1 (Fig. 3A). Leukocyte numbers continued to increase in the lungs of WT mice through weeks 2 and 3. Leukocyte numbers also increased in the lungs of IL-4 KO and IL-10 KO mice but not to the same magnitude as that observed in WT mice (Fig. 3A). At weeks 2 and 3, leukocyte recruitment into the lungs of IL-4 KO and IL-10 KO mice was significantly less than that in WT mice (Fig. 3A). At week 3, the pulmonary leukocytic infiltrate in WT mice was extensive whereas the inflammatory response was largely localized to a small number of discreet foci in the lungs of IL-4 KO and IL-10 KO mice (Fig. 3B). Thus, all three groups of mice developed inflammatory responses in their lungs, but the magnitude of the response was significantly less in the absence of IL-4 or IL-10.



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  		FIGURE 3. Leukocyte recruitment into the lungs of C. neoformans infected mice. A, Total lung leukocytes were isolated from individual lungs and the leukocyte recruitment represented as the difference between infected and uninfected mice. *, p < 0.05, comparing WT vs IL-10 KO or IL-4 KO mice; n = 11–12 mice/group/time point for weeks 1 and 2 and 6 mice/group/time point for week 3. B, Low-power photomicrographs of the inflammatory infiltrates in the lungs at 3 wk postinfection. H&E magnification, x50.

 
Next, the cellular composition of the leukocyte infiltrate was analyzed. Leukocytes from total lung digests were identified by Wright-Giemsa staining and flow cytometry. There were transient differences in lymphocyte recruitment among the three groups of mice at weeks 1–3 (Fig. 4). Also, at weeks 1 and 2, there were transient differences in the recruitment of macrophages and neutrophils into the lungs of IL-4 KO and IL-10 KO mice. At week 3, there were significantly more macrophages and neutrophils in WT than IL-4 KO and IL-10 KO mice (Fig. 4). The most distinguishing feature of the inflammatory response in WT vs IL-4 KO vs IL-10 KO mice was the presence of eosinophils in the leukocyte infiltrate. WT mice had significantly more eosinophils in the lungs at every time point examined (Fig. 4). In contrast, pulmonary eosinophilia was virtually absent in IL-4 KO and IL-10 KO mice. Therefore, in the absence of IL-4 or IL-10, an allergic type inflammatory response did not develop in C. neoformans-infected C57BL/6 mice, indicating that both the type and magnitude of the pulmonary inflammatory response was regulated by IL-4 and IL-10.



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  		FIGURE 4. Leukocyte subset recruitment into the lungs of WT, IL-10 KO, and IL-4 KO mice. Samples of leukocyte suspensions were cytospun onto slides and stained in Wright-Giemsa for visual quantification of macrophages, neutrophils, and eosinophils. CD4, CD8, and B cells were acquired by flow cytometry. *, p < 0.05, comparing C57BL/6 vs IL-10 KO or IL-4 KO mice using ANOVA test; n = 5–7 mice/group/time point.

 
Finally, the inflammatory response was analyzed histologically at week 3. It is evident in the photomicrographs that the magnitude of the pulmonary inflammatory response was significantly greater in WT mice (Fig. 5, B, F, and J) than in IL-4 KO (Fig. 5, C, G, and K) or IL-10 KO mice (Fig. 5, D, H, and L). The inflammatory infiltrate in the lungs of WT mice consisted mainly of neutrophils, macrophages and eosinophils and mirrored the quantitative analysis of whole lung enzymatic digests (Figs. 3 and 4). The limited inflammation in IL-4 KO and IL-10 KO mice largely consisted of mononuclear cells. Slightly enhanced pulmonary fibrosis was evident in sections from WT mice (Fig. 5, J–L). However, the most striking histological difference was the lack of goblet cell metaplasia in IL-4 KO and IL-10 KO mice compared with WT mice (Fig. 5, E–H), which correlated with the decreased pulmonary IL-13 levels in IL-4 KO and IL-10 KO mice (Fig. 6). Goblet cell metaplasia is a significant feature of allergic airway disease and the absence of goblet cell metaplasia in IL-4 KO and IL-10 KO mice provides additional evidence that these cytokines are crucial for driving the allergic response during C. neoformans infection in C57BL/6 mice. In addition, because pulmonary clearance of the fungal infection is greater in IL-4 KO and IL-10 KO than WT mice, these data also indicate that the type rather than simply the magnitude of the pulmonary inflammatory response is a critical factor for clearing the infection.



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  		FIGURE 5. Photomicrographs of C. neoformans infected lungs at week 3 postinfection. Lung tissue sections from uninfected WT mice (A, E, and I), C. neoformans-infected WT (B, F, and J), IL-4 KO (C, G, and K), and IL-10 KO (D, H, and L) mice. H&E (A–D), periodic acid-Schiff (E–H) stain, to stain mucus and mucus-secreting goblet cells, with a hematoxylin counterstain (stains goblet cells bright pink), and Masson’s trichrome (I–L) (collagen stains blue). Magnification, x250.

 


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  		FIGURE 6. T1- and T2-type cytokine production by lung leukocytes. Total lung leukocytes were obtained 1 and 2 wk postinfection and cultured for 24 h at 5 x 106 cells/ml. Lung leukocytes were cultured in the absence of exogenous stimuli (No Ag) or in the presence of HKC at 1 x 107/ml. Supernatants were harvested and cytokine levels measured by ELISA. *, p < 0.05 for WT vs IL-10 KO or vs IL-4 KO; n = 11–12 mice/group/time point for weeks 1 and 2.

 
Characterization of the T1/T2 immune response in the lung and LALN

To analyze the T1/T2 polarization of the response, IFN-{gamma}, IL-4, IL-5, and IL-13 production by lung leukocytes was analyzed at weeks 1 and 2 postinfection. Total lung leukocytes were isolated and cultured. To control for possible differences in the amount of cryptococci (Ag) between the different groups, cultures were set up with either no additional Ag or with 107 HKC (>20:1 ratio of exogenous to endogenous cryptococci). Cytokines were measured in the supernatants after 24 h of culture. Lung leukocytes from WT, IL-4 KO, and IL-10 KO mice all produced significant levels of IFN-{gamma} in the presence or absence of additional Ag (Fig. 6). In contrast, lung leukocyte production of IL-4, IL-5, and IL-13 in the absence of exogenous Ag was significantly lower in both IL-4 KO and IL-10 KO mice compared with WT mice (Fig. 6). In the presence of exogenous Ag in the cultures, IL-5 and IL-13 were significantly lower at week 2 in both IL-4 KO and IL-10 KO mice compared with WT mice (Fig. 6). HKC-stimulated IL-4 levels from IL-10 KO lung leukocytes were significantly lower at week 1 and did not increase at week 2. HKC-stimulated IL-13 levels were significantly lower at week 1 in the IL-4 KO lung leukocyte cultures. In summary, the production of IFN-{gamma} or T2 cytokines was not augmented in IL-10 KO mice. The production of T2 cytokines by lung leukocytes from IL-4 KO and IL-10 KO mice was lower than that from WT mice and the addition of exogenous Ag did not completely abrogate the differences.

We next analyzed the draining lymph nodes (LALN) for evidence of changes in T1 vs T2 polarization. Despite the presence of low numbers of cryptococci in the LALN (Fig. 1), these cells did not produce cytokines in vitro unless restimulated with HKC (Fig. 7). At both weeks 1 and 2, LALN cells from WT mice produced IFN-{gamma}, IL-5, and IL-13 (Fig. 7). Production of IL-4 was minimal. There was no difference in IFN-{gamma}, IL-4, IL-5, or IL-13 production by LALN cells from WT or IL-10 KO mice (Fig. 7). In contrast to LALN cells from IL-10 KO mice, LALN cells from IL-4 KO mice produced significantly lower levels of IL-13 and IL-4 (Fig. 7) IL-5 production was also lower at week 2. In addition, serum IgE levels were at uninfected levels in IL-4 KO mice but were significantly elevated in both WT and IL-10 KO mice (Fig. 8). Thus, IL-4 KO mice do not appear to develop the systemic (LALN and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remain unaltered in IL-10 KO mice whereas the T2 response in the lungs fails to develop.



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  		FIGURE 7. T1 and T2 cytokine production by lung-associated lymph node cells. Lymph node cells were obtained (described in Materials and Methods) 1 and 2 wk postinfection and cultured for 24 h at 5 x 106 cells/ml. Cells were cultured in the absence of exogenous stimuli (No Ag) or in the presence of HKC at 1 x 107/ml. Supernatants were harvested and cytokine levels measured by ELISA. *, p < 0.05 for WT vs IL-10 KO or vs IL-4 KO; n = 11–12 mice/group/time point.

 


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  		FIGURE 8. Total serum IgE levels in C. neoformans-infected mice. Total serum IgE levels were determined by ELISA. Dotted line represents IgE levels for uninfected mice. *, p < 0.05, comparing WT vs IL-10 KO or IL-4 KO mice; n = 11–12 mice/group/time point for weeks 1 and 2 and 6 mice/group/time point for week 3.

 
We also examined whether TNF-{alpha} and IL-12 production by lung leukocytes was altered in IL-4 KO and IL-10 KO mice. At week 2, TNF-{alpha} levels were higher in lung leukocyte cultures from IL-4 KO mice compared with WT mice. But there was no difference in lung leukocyte production of IL-12 or IL-10 between WT and IL-4 KO mice (Fig. 9). However, lung leukocyte production of TNF-{alpha} and IL-12 was significantly elevated and IL-10 absent at weeks 1 and 2 in IL-10 KO mice (Fig. 9). TNF-{alpha} production was also significantly elevated in LALN leukocytes at week 2 (Fig. 9). Thus, in the absence of IL-10, local (pulmonary) TNF-{alpha} and IL-12 production by lung leukocytes is up-regulated, favoring the manifestation of a T1 response in the lungs.



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  		FIGURE 9. Inflammatory cytokine production by lung leukocytes and lymph node cells. Lung leukocytes (A) and LALN (B) were obtained 1 and 2 wk postinfection and cultured for 24 h at 5 x 106 cells/ml. Cells were cultured in the absence of exogenous stimuli (No Ag) or in the presence of HKC at 1 x 107/ml. Supernatants were harvested and cytokine levels measured by ELISA. *, p < 0.05 for WT vs IL-10 KO or vs IL-4 KO; n = 11–12 mice/group/time point for weeks 1 and 2 and 6 mice/group/time point for week 3.

 
Role of IFN-{gamma} in controlling C. neoformans growth in the lungs

Because IFN-{gamma} production by LALN or lung leukocytes did not significantly change in the absence of IL-4 or IL-10, even though clearance was enhanced, this raised the issue of whether IFN-{gamma} plays a role in controlling the growth of C. neoformans in the lungs of C57BL/6 mice. To address this question, IFN-{gamma} KO C57BL/6 mice were infected and the pulmonary burden at 3 wk postinfection was compared with WT mice. As illustrated in Fig. 10, the pulmonary burden in WT mice was high but the number of organisms in the lungs of IFN-{gamma} KO mice at this time point was significantly higher. Thus, despite production of potentially antagonistic cytokines during cryptococcal infection in C57BL/6 mice, IFN-{gamma} is required to limit the extent of the infection with pulmonary CFU levels being 50-fold higher in IFN-{gamma} KO C57BL/6 mice.



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  		FIGURE 10. Pulmonary levels of C. neoformans in WT and IFN-{gamma} KO mice at week 3 postinfection following intratracheal inoculation (104 CFU). *, p < 0.05, comparing WT vs IFN-{gamma} KO mice; n = 6 mice/group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies are the first to focus on the role of IL-4 and IL-10 in the context of a chronic APBM caused by C. neoformans. Both IL-4 KO and IL-10 KO mice have enhanced pulmonary clearance of C. neoformans compared with WT C57BL/6 mice. It has been demonstrated previously that IL-10 KO mice have increased survival following i.v. inoculation of C. neoformans (strain 24067) and pulmonary infection with C. neoformans NU-2 (35, 36). In contrast, there was not a difference in survival between WT and IL-4 KO mice in these studies (35, 37). Interestingly, delayed-type hypersensitivity responses were reduced in IL-4 KO mice following infection with the less virulent C. neoformans strain 184A but splenic T1 cytokine responses were increased (37). IL-5 and IL-10 production by splenocytes was down-regulated in IL-4 KO C57BL/6 mice. However, in i.v. infection with C. neoformans 1841 in B6 x 129F2 mice, there was a significant enhancement of survival in IL-4 KO mice (20). Similar findings were reported using anti-IL-4 mAb to treat CDF1 mice infected intratracheally with C. neoformans YC-11 (15). To put all these studies in the context of our current results and model, it needs to be noted that none of the animal model systems just described are true "chronic" pulmonary infections; in other words, all of the control animals still die. In contrast, C57BL/6 mice (6–8 wk at time of infection) infected with C. neoformans strain 24067 can survive >12 wk while harboring a stable C. neoformans burden on the lungs of 106–107 CFU (3, 4). Thus, the role of IL-4 is going to be influenced by the cytokine milieu and virulence of the cryptococcal strain. The major findings of our current study are that in ABPM caused by C. neoformans: 1) IL-4 and IL-10 promote the development of a T2 response (eosinophilia, IL-4, IL-5, IL-13) in the lungs and the subsequent chronic pulmonary infection; 2) IFN-{gamma} induction is independent of the influence of IL-4 and IL-10; and 3) IL-4 promotes T2 type anti-cryptococcal immunity systemically and at the site of infection whereas IL-10 only promotes a T2 anti-cryptococcal response locally (lungs).

C. neoformans-infected C57BL/6 mice produced both T1 and T2 cytokines in their lungs and LALN. Previous studies have shown that LALN and lung cells from C. neoformans-infected C57BL/6 mice fail to produce IL-2 and secrete very low levels of IFN-{gamma} when compared with resistant mice (3). Our studies showed that C57BL/6 lung leukocytes and lymph node cells secrete significant levels of IFN-{gamma}. However, despite the secretion of IFN-{gamma}, C57BL/6 mice develop T2 responses in their lungs and lymph nodes. Also, previous data had shown that C57BL/6 mice produce elevated levels of IL-5 and develop chronic eosinophilia in their lungs (3, 4). The studies presented in this report demonstrate that lung leukocytes from C57BL/6 mice also produce high levels of IL-4 and IL-13 and the immune response also includes high serum IgE levels. Lung leukocytes from C. neoformans-infected C57BL/6 mice also secrete IL-12 and TNF-{alpha}. Thus, lung and LALN leukocytes from C57BL/6 mice secrete IFN-{gamma}, IL-12, and TNF-{alpha} as well as IL-4, IL-5, and IL-13, creating a mixed T1/T2 environment in the lungs and lung draining lymph nodes. However, the T1 and T2 cytokines do not appear to be equivalent and the inflammatory response that develops contains a high percentage of eosinophils, similar to that observed in asthma models (38). The end result is a chronic pulmonary fungal infection.

We noted in our studies that both IL-12 and TNF-{alpha} production by lung leukocytes was enhanced in IL-10 KO mice. IL-10 can directly down-regulate the production of a number of cytokines by myeloid and lymphoid leukocytes following exposure to C. neoformans, including TNF-{alpha} and IL-12 (39, 40, 41, 42, 43). The polysaccharide capsule of C. neoformans (glucuronoxylomannan) is a potent inducer of IL-10 from leukocytes (40, 42, 44, 45). In addition, IL-10 is normally produced during inflammatory responses as one mechanism to regulate inflammatory responses and prevent over-exuberant inflammation such as is seen in inflammatory bowel disease (46). IL-12 and TNF-{alpha} are required for the development of protective pulmonary T1 responses to C. neoformans (9, 11, 14, 16, 20, 21, 22, 47). Thus, the high levels of IL-12 and TNF-{alpha} in C. neoformans-infected IL-10 KO C57BL/6 mice is consistent with the strong T1 response in these mice.

Another observation from these studies is that strong polarization of the cell-mediated response to C. neoformans does not appear to occur in the lymph nodes. Rather, early polarization of the response appears to occur largely at the site of infection, the lungs, despite the presence of cryptococci in the draining lymph nodes. For the most part, both T1 and T2 responses coexist in the LALN of C. neoformans-infected C57BL/6 mice, similar to the LALN from B6 x 129F2 mice (13). Both IL-4 KO and IL-10 KO C57BL/6 mice have a significantly diminished allergic (T2) response in the lungs and are able to begin to clear the infection. At this stage of the investigation, we are not sure of the cellular sources of the cytokines in the LALN vs lungs or whether they are different in IL-4 KO vs IL-10 KO vs WT. However, these studies demonstrate that the polarization of the cellular response in the LALN vs lungs is different. Is it possible that T cells polarize in the LALN and then immediately emigrate to the lungs? We cannot formally exclude this possibility in this model, but in other studies from our laboratory that have addressed this question during protective (T1) immunity to C. neoformans, it is clear that CD4 T cell polarization does not occur in the LALN, blood or spleen but is evident in the lungs.4 Our studies indicate that IL-4 also promotes the development of the T2 response in the LALN whereas IL-10 does not play any role in regulating the response in the LALN during an allergic response to C. neoformans.

Surprisingly, despite the higher production of TNF-{alpha} and IL-12 in the lungs of IL-10 KO mice, and the reduction of T2 cytokines in both IL-4 KO and IL-10 KO mice, the levels of IFN-{gamma} were not significantly enhanced in these mice. One possibility is that, in the absence of IL-4 and IL-10, the levels of IFN-{gamma} are sufficient to drive protective immunity and other non-IL-4 or non-IL-10 mechanisms keep an overexuberant IFN-{gamma} response from developing. In other studies, it appears that a non-T cell source of IFN-{gamma} is responsible for the low-level protection of C57BL/6 during pulmonary cryptococcosis.5 The results from the current studies indicate that the production of IFN-{gamma} is independent of regulation by IL-4 and IL-10. Furthermore, IFN-{gamma} is required for control of the infection. In studies of allergic bronchopulmonary cryptococcosis using IFN-{gamma} KO C57BL/6 mice, it appears that IFN-{gamma} production is not required for the development of T2 cytokine producing cells in the lungs and LALN and T2 cytokine (IL-4, IL-5, IL-13) production was enhanced at a number of time points in IFN-{gamma} KO compared with WT C57BL/6 mice.5

Although the in vivo anti-inflammatory properties of IL-10 are fairly consistent throughout the literature, our studies demonstrate that the fungal agent may be an important consideration in understanding the role of IL-10 in ABPM. Pulmonary cryptococcosis with strain 24067 in C57BL/6 mice shares many features with murine models of allergic bronchopulmonary aspergillosis. These include high IgE, elevated peripheral blood and lung eosinophils, pulmonary inflammation, elevated levels of IL-4, IL-5, and IL-13, production of IFN-{gamma}, pulmonary fibrosis, goblet cell hyperplasia and chronic fungal colonization/persistence (3, 4, 48, 49, 50, 51). In murine models of allergic bronchopulmonary aspergillosis, the T2 cytokines IL-4, IL-5, and IL-13 are required for these pathologic features of the host response (48, 49, 50, 51). Both IL-4 and IL-10 can play significant roles in regulating IL-4, IL-5, IL-10 and IL-13 responses to purified allergens (33, 34). In a murine model of Aspergillus Ag-induced inflammation, the allergic lung response is similar in intranasally primed IL-10 KO and WT C57BL/6 mice (52). However, pulmonary eosinophilia is heightened in IL-10 KO outbred mice primed intranasally or in IL-10 KO C57BL/6 mice if the mice are primed i.p. before Ag challenge (52). The pulmonary allergic response was not diminished in any of these settings, in stark contrast to the allergic response to live challenge with the yeast cryptococcus. Thus, in contrast to Aspergillus Ag-induced pulmonary inflammation, C. neoformans causes an allergic bronchopulmonary inflammatory response (T2) that is augmented by IL-10 production.

Similar to observations in other models of allergic airway disease (38), it appears that the inflammatory response to C. neoformans in WT C57BL/6 mice contains both T1 and T2 elements and our studies suggest that the T2 response does not antagonize the T1-mediated inflammatory response. Although speculative at this point in our studies, it appears that the macrophages are altered in this T2 environment, making them less able to control the growth of the organisms. Previous studies have demonstrated that the production of NO, but not iNOS, is deficient in WT mice (53). Studies of macrophages activated in high IL-4/IL-13 environments (alternatively activated macrophages) indicate that these macrophages produce higher levels of arginase and are poor at killing intracellular microbes (54). In WT C57BL/6 mice, high intramacrophage burdens of yeast are evident (4), consistent with the presence of alternatively activated macrophages. In IL-4 and IL-10 KO mice, there is little histologic evidence for the generation of alternatively activated macrophages, suggesting that the IFN-{gamma} produced in the absence of antagonizing signals (IL-4, IL-10, IL-13) generates macrophages that are very efficient at killing C. neoformans. It is our hypothesis that this is why the overall magnitude of the inflammatory response is lower in IL-4 and IL-10 KO mice but clearance is greater. Thus, our studies indicate that it is not the magnitude of the inflammatory response that is important, it is the type of response.

Chronic infections can result from an imbalance in the ratio of T1 to T2 cytokines produced against a pathogen. However, recent studies also indicate that suppression of a protective immune response can be explained by the induction of regulatory T cells by the microbe. Regulatory T cells are immunosuppressive CD4+ T cells with cytokine profiles distinct from either type 1 (Th1) or type 2 (Th2) T cells (55). Regulatory T cells play an essential role in the maintenance of self-tolerance. Also, they have recently been described as a possible mechanism used by pathogens to suppress protective immune responses (56, 57, 58). Regulatory T1 T cells are one of the subsets of regulatory T cells that express the markers CCR5, T1-ST2, CD25, CD45RO, and CD45RBlow. Most regulatory T1 T cells exclusively secrete high levels of IL-10, which they use as the suppressor mechanism, but some can also secrete IL-5 and TGF-{beta}. They produce little or no IL-2, thereby proliferating poorly, and produce no IL-4 or IFN-{gamma}. Studies performed in the pulmonary pathogen Bordetella pertussis, have shown that regulatory T1 T cells can prevent the development of Th1 responses through the production of IL-10, introducing a new strategy used by pathogens in the respiratory tract to prolong survival of a pathogen (56). Such a mechanism may occur in allergic bronchopulmonary cryptococcosis; however, IL-10 seems to be playing a positive role in the pulmonary T2 response rather than a negative role in the T1 response because production of IFN-{gamma} by lung leukocytes appears to be unaltered in IL-10 KO mice (however, the cellular source of IFN-{gamma} may be different in WT vs IL-10 KO mice).


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants from the National Institutes of Health R01-HL065912 and R01-AI059201 (to G.B.H.), R01-HL051082 (to G.B.T.). G.B.H. was also supported in part by a New Investigator Award in Molecular Pathogenic Mycology from the Burroughs-Wellcome Fund. Y.H. was supported in part by a Rackham Graduate Fellowship from the University of Michigan, Ann Arbor, MI. Back

2 Address correspondence and reprint requests to Dr. Gary B. Huffnagle, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, 6301 Medical Sciences Research Building III, Box 0642, University of Michigan Medical School, Ann Arbor, MI 48109-0642. E-mail address: ghuff{at}umich.edu Back

3 Abbreviations used in this paper: ABPM, allergic bronchopulmonary mycosis; LALN, lung-associated lymph nodes; KO, knockout; WT, wild type; HKC, heat-killed C. neoformans. Back

4 D. M. Lindell, T. A. Moore, R. A. McDonald, G. B. Toews, and G. B. Huffnagle, Polarization of CD4+ T cells occurs in non-lymphoid tissues. Submitted for publication. Back

5 S. A. Arora, Y. Hernandez, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, and G. B. Huffnagle, Role of IFN-{gamma} in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis. Submitted for publication. Back

Received for publication January 23, 2004. Accepted for publication November 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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U. Muller, W. Stenzel, G. Kohler, C. Werner, T. Polte, G. Hansen, N. Schutze, R. K. Straubinger, M. Blessing, A. N. J. McKenzie, et al.
IL-13 Induces Disease-Promoting Type 2 Cytokines, Alternatively Activated Macrophages and Allergic Inflammation during Pulmonary Infection of Mice with Cryptococcus neoformans
J. Immunol., October 15, 2007; 179(8): 5367 - 5377.
[Abstract] [Full Text] [PDF]

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 Infect. Immun.Home page
J. E. Milam, A. C. Herring-Palmer, R. Pandrangi, R. A. McDonald, G. B. Huffnagle, and G. B. Toews
Modulation of the Pulmonary Type 2 T-Cell Response to Cryptococcus neoformans by Intratracheal Delivery of a Tumor Necrosis Factor Alpha-Expressing Adenoviral Vector
Infect. Immun., October 1, 2007; 75(10): 4951 - 4958.
[Abstract] [Full Text] [PDF]

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 Am. J. Pathol.Home page
G.-H. Chen, M. A. Olszewski, R. A. McDonald, J. C. Wells, R. Paine III, G. B. Huffnagle, and G. B. Toews
Role of Granulocyte Macrophage Colony-Stimulating Factor in Host Defense Against Pulmonary Cryptococcus neoformans Infection during Murine Allergi