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

This information is current as Yadira Hernandez, Shikha Arora, John R. Erb-Downward, of October 1, 2021. Roderick A. McDonald, Galen B. Toews and Gary B. Huffnagle J Immunol 2005; 174:1027-1036; ; doi: 10.4049/jimmunol.174.2.1027 http://www.jimmunol.org/content/174/2/1027 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

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

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-␥ levels remained the same. TNF-␣ and IL-12 production by lung leukocytes was Downloaded from 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. The Journal of Immunology, 2005, 174: 1027–1036. http://www.jimmunol.org/

he fungal pathogen Cryptococcus neoformans enters the involves the up-regulation of T2 immunity. C. neoformans chronic body through inhalation and establishes a primary pulmo- infection stays primarily localized in the lungs of C57BL/6 mice, T nary infection. Inbred mouse models of pulmonary cryp- with minimal dissemination to extrapulmonary sites such as the tococcosis indicate a genetic component to the response. CBA/J spleen and the CNS. When compared with CBA/J or C.B-17 mice, and C.B-17 mice clear the infection faster than BALB/c mice. susceptibility of C57BL/6 mice does not correlate with the level of C57BL/6 mice can develop a chronic pulmonary infection (1–4). inflammation at the site of infection, but does correlate with high Clearance of C. neoformans in resistant hosts involves the devel- ␥ levels of IL-5 secretion, low levels of IFN- , and low levels of by guest on October 1, 2021 opment of T1 cell-mediated immunity (5–9). T cells recruit and IL-2 production (2–4). Elevated levels of IL-5 in the lungs of activate phagocytic effector cells, such as macrophages, against C. C57BL/6 mice promote the development of pulmonary eosino- neoformans inducing a granulomatous reaction and the develop- philia, which results in eosinophil-mediated tissue damage in the ment of delayed-type hypersensitivity responses (10). Many cyto- lungs, including deposition of eosinophilic YM1 crystals (4, 24). kines, mainly T1 cytokines, have been shown to play important The level of susceptibility in C. neoformans infection correlates roles in clearance of C. neoformans. Among these cytokines are with the number of eosinophils infiltrating the lungs (4). Suscep- ␥ ␣ IFN- , IL-2, IL-12, IL-18, TNF- , and IL-15, and the CC chemo- tible C57BL/6 mice have a large number of eosinophils in their kine ligands type 2 and type 3 (3, 9, 11–22). lungs, moderately resistant BALB/c mice have transient influx of Chronic fungal infections can develop when the T1/T2 balance eosinophils, and highly resistant CBA/J mice have only a few eo- of cellular immunity is shifted away from T1 toward T2 immune sinophils in their lungs (4). Thus, low dose infection of 6- to 8-wk- responses (23). C57BL/6 mice, 6- to 8-wk-old at the time of in- old C57BL/6 mice with C. neoformans strain 24067 produces a fection, develop a nonresolving pulmonary fungal infection, which chronic allergic bronchopulmonary mycosis (ABPM).3 This model has been used to address the role of immunomodulatory agents such as OX40, Mycobacterium bacillus Calmette-Gue´rin, ␣-galac- *Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and †Department of Microbiology and Immunology, University of Michigan Medical tosylceramide (a CD1 ligand), IL-5 antagonists and anti-capsular School, Ann Arbor, MI 48109 Abs in addition to antifungal drugs in modulating immunity and Received for publication January 23, 2004. Accepted for publication November promoting protective host responses (25–32). Because both IL-4 2, 2004. and IL-10 can play significant regulatory roles in T2 responses to The costs of publication of this article were defrayed in part by the payment of page purified allergens (33, 34), our objective was to investigate the role charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of IL-4 and IL-10 in the development and manifestation of the T2 1 This work was supported by Grants from the National Institutes of Health R01- response to C. neoformans in the lungs and lung-associated lymph HL065912 and R01-AI059201 (to G.B.H.), R01-HL051082 (to G.B.T.). G.B.H. was nodes (LALN) in this model of ABPM. also supported in part by a New Investigator Award in Molecular Pathogenic My- cology from the Burroughs-Wellcome Fund. Y.H. was supported in part by a Rack- ham Graduate Fellowship from the University of Michigan, Ann Arbor, MI. 2 Address correspondence and reprint requests to Dr. Gary B. Huffnagle, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, 6301 Med- 3 Abbreviations used in this paper: ABPM, allergic bronchopulmonary mycosis; ical Sciences Research Building III, Box 0642, University of Michigan Medical LALN, lung-associated lymph nodes; KO, knockout; WT, wild type; HKC, heat- School, Ann Arbor, MI 48109-0642. E-mail address: [email protected] killed C. neoformans.

Materials and Methods plete medium. Total lung leukocyte numbers were assessed in the presence Mice of trypan blue using a hemocytometer. Female IL-4 knockout (KO), IL-10 KO, and wild-type (WT) mice on a Lung leukocyte subsets Ϯ C57BL/6 background (16 2 g) were obtained from The Jackson Labo- Macrophages, neutrophils, and eosinophils were visually counted in ratory. Mice were 6- to 8-wk-old at the time of infection. Mice were housed Wright-Giemsa-stained samples of lung cell suspensions cytospun onto in sterilized cages covered with a filter top. Food and water were given ad glass slides (Thermo Shandon Cytospin). For Wright-Giemsa staining, the libitum. The mice were maintained by the Unit for Laboratory Animal slides were fixed for 2 min with a one-step methanol-based Wright-Giemsa Medicine, University of Michigan, in accordance with regulations ap- stain (Harleco; EM Diagnostics Systems) followed by steps two and three proved by the University of Michigan Committee on the Use and Care of of the Diff-Quik whole blood stain kit (Diff-Quik; Baxter Scientific Prod- Animals. ucts). A total of 200–300 cells were counted from randomly chosen high C. neoformans culture power microscope fields for each sample. The percentage of a leukocyte subset was multiplied by the total number of leukocytes to give the abso- C. neoformans strain 24067 (52D) was obtained from the American Type lute number of that type of leukocyte in the sample. Culture Collection. For injection, yeast were grown to stationary phase Numbers of B, CD4, and CD8 T cells were determined by flow cytom- 5 (48–72 h) at 37°C in Sabouraud dextrose broth (1% neopeptone and 2% etry. Lung leukocytes (5 ϫ 10 ) were incubated for 30 min on ice in a total ␮ dextrose; Difco) on a shaker. The cultures were then washed in nonpyro- volume of 120 l of staining buffer (FA buffer; Difco), 0.1% NaN3, and 1% genic saline, counted on a hemocytometer, and diluted to 3.3 ϫ 105 FCS. Each sample was incubated with 1 ␮g of the respective FITC- or CFU/ml in sterile nonpyrogenic saline. PE-labeled mAb (BD Pharmingen), or isotype-matched rat IgG. The samples were washed in staining buffer and fixed in 1% paraformaldehyde Surgical intratracheal inoculation (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 Mice were anesthetized by i.p. injection of pentobarbital (0.074 mg/g lymphocyte subset was multiplied by the total number of leukocytes to give Downloaded from weight of mouse) and restrained on a small surgical board. A small incision the absolute number of that type of lymphocyte in the sample. 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 Histology filled with diluted C. neoformans culture. The needle was inserted into the trachea, and 30 ␮l of inoculum (104 CFU) were dispensed into the lungs. Lungs were fixed by inflation with 10% neutral-buffered formalin. After ␮ The needle was removed and the skin closed with cyanoacrylate adhesive. paraffin embedding, 5- m sections were cut and stained with H&E, peri- The mice recovered with minimal visible trauma. odic acid-Schiff, to stain mucus and mucus-secreting goblet cells, or Mas-

son’s trichrome (collagen deposition stains blue). http://www.jimmunol.org/ CFU assay Preparation of lymph nodes For lung and LALN CFU, small aliquots were collected from lung digests or lymph node suspensions, respectively (described below). Aliquots of the LALN from two to three mice were pooled and processed into a cell sus- lungs and lymph nodes were plated out on Sabouraud dextrose agar plates pension by gently passing tissues through nylon mesh. Cells were then in duplicate 10-fold dilutions and incubated at room temperature. C. neo- washed and resuspended in complete RPMI 1640 medium. Total viable cell formans colonies were counted 2–3 days later, and the number of CFU per numbers were assessed by trypan blue exclusion and counted on a organ was calculated. hemocytometer. Induction of T cell deﬁciency Lung leukocyte and lymph node cultures

ϫ 6 by guest on October 1, 2021 Mice were treated with 300 ␮g of anti-CD4 plus 300 ␮g of anti-CD8 mAb Isolated lung leukocytes or lymph node cells (5 10 /ml) were cultured (GK1.5 and YTS 169.4, respectively) or anti-CD4 alone or anti-CD8 alone for 24 h in 24-well plates with 2 ml of complete RPMI 1640 medium at on day 0 of the infection and boosted with 100 ␮g of each mAb at days 7 37°C and 5% CO2 with or without additional stimulus. Cultures were sup- ϫ 7 and 14. T cell depletion was analyzed by ﬂow cytometry of spleen cells. plied with heat-killed C. neoformans (HKC) at 1 10 /ml where indi- Ͼ ϩ Ͼ ϩ cated. Positive controls were performed by incubating the cells in the pres- Depletion was 99% for CD4 T cells and 95% for CD8 T cells (data ␮ not shown). ence of 5 g/ml Con A. Lung leukocyte isolation Cytokine production Culture supernatants were harvested at 24 h and assayed for IFN-␥, IL-4, Individual lungs were excised, minced, and enzymatically digested for 30 IL-5, IL-13, TNF-␣, IL-12, and IL-10 production by sandwich ELISA min in 15 ml of digestion buffer (RPMI 1640, 5% FCS, antibiotics, 1 using the manufacturer’s instructions supplied with the cytokine-speciﬁc mg/ml collagenase, and 30 ␮g/ml DNase). The cell suspension and undi- kits (BD Pharmingen and R&D Systems). gested 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 Total serum IgE 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 Serum was obtained by tail vein bleed of the mice and spinning the blood 7.4). Ten-fold excess of medium was added to return the solution to iso- to obtain the serum. Serum samples were then assayed using an IgE-spe- tonicity. The isolated leukocytes were repelleted and resuspended in com- ciﬁc sandwich ELISA (BD Pharmingen).

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 ,ء .removed, processed and plated for CFU p Ͻ 0.05, comparing WT vs IL-10 KO or IL-4 KO mice; n ϭ 11–12 mice/group/time point for weeks 1 and 2 and 6 mice/group/ time point for week 3. The Journal of Immunology 1029

Statistics Statistical significance was calculated using ANOVA test (least significant difference posthoc) with significance being p Ͻ 0.05 for comparison between WT and IL-4 KO or WT and IL-10 KO. All values are reported as mean Ϯ SEM for each group of pooled data derived from two to four experiments. Results 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 (Ͼ log 106 CFU) through weeks 2–3 (Fig. 1A). This is consistent with previously published studies demonstrating that C57BL/6 mice are FIGURE 2. Effect of T cell depletion on C. neoformans pulmonary Downloaded from unable to clear a pulmonary C. neoformans infection (3, 4). In growth. WT and KO mice were depleted in vivo of CD4, CD8, or both contrast, IL-4 KO and IL-10 KO mice began to clear the infection CD4 and CD8 T cells using mAbs. Lungs were harvested at week 3 and p Ͻ 0.05 comparing ,ء .during weeks 2–3. By week 3, the number of cryptococci in the aliquots from lung digests were plated for CFU lungs of IL-4 KO and IL-10 KO mice was 500- to 1000-fold lower nondepleted to depleted mice within the same genetic background of mice than in WT mice (Fig. 1). Thus, in contrast to the chronic infection (WT, IL-4 KO, or IL-10 KO); n ϭ 3–8 mice/group. in WT mice, pulmonary clearance of C. neoformans was significantly greater in IL-4 KO and IL-10 KO mice, suggesting that http://www.jimmunol.org/ T cells did not significantly alter the pulmonary cryptococcal bur- production of IL-4 and IL-10 can promote the development of the den in WT mice (Fig. 2). In contrast, depletion of both CD4 and chronic pulmonary infection. These data also demonstrate that ef- CD8 T cells abrogated pulmonary clearance in both IL-4 KO and fector mechanisms are not inherently defective in C57BL/6 mice IL-10 KO mice (Fig. 2). Pulmonary CFU in CD4/CD8 T cell- because IL-4 KO and IL-10 KO C57BL/6 mice are able to clear depleted KO mice was identical with that in WT mice (Fig. 2). the infection. Depletion of either CD4 or CD8 T cells alone in both IL-4 KO and 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 by guest on October 1, 2021 pathogen-free housing, LALN are undetectable (Ͻ104 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 develop- FIGURE 3. Leukocyte recruitment into the lungs of C. neoformans infected mice. A, Total lung leukocytes were isolated from individual lungs ment of a T cell-mediated immune response. Mice were rendered and the leukocyte recruitment represented as the difference between in- p Ͻ 0.05, comparing WT vs IL-10 KO or ,ء .CD4 and CD8 T cell deficient by treatment with anti-CD4 and fected and uninfected mice anti-CD8 mAbs every 7–8 days beginning at the onset of infection. IL-4 KO mice; n ϭ 11–12 mice/group/time point for weeks 1 and 2 and 6 CD4 and CD8 T cell depletion was Ͼ95% in these animals (data mice/group/time point for week 3. B, Low-power photomicrographs of the not shown). T cell replete WT mice were unable to clear C. neo- inflammatory infiltrates in the lungs at 3 wk postinfection. H&E magnifi- formans from their lungs (Fig. 2) and depletion of CD4 and CD8 cation, ϫ50. 1030 IL-4 AND IL-10 IN T2 IMMUNITY DURING ABPM

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. 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 Downloaded from 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 ex- tensive whereas the inflammatory response was largely localized http://www.jimmunol.org/ 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. 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 by guest on October 1, 2021 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 FIGURE 4. Leukocyte subset recruitment into the lungs of WT, IL-10 every time point examined (Fig. 4). In contrast, pulmonary eosino- KO, and IL-4 KO mice. Samples of leukocyte suspensions were cytospun philia was virtually absent in IL-4 KO and IL-10 KO mice. Therefore, onto slides and stained in Wright-Giemsa for visual quantification of macro- in the absence of IL-4 or IL-10, an allergic type inflammatory re- phages, neutrophils, and eosinophils. CD4, CD8, and B cells were acquired by p Ͻ 0.05, comparing C57BL/6 vs IL-10 KO or IL-4 KO ,ء .sponse did not develop in C. neoformans-infected C57BL/6 mice, flow cytometry ϭ indicating that both the type and magnitude of the pulmonary inflam- mice using ANOVA test; n 5–7 mice/group/time point. matory response was regulated by IL-4 and IL-10. Finally, the inflammatory response was analyzed histologically at week 3. It is evident in the photomicrographs that the magnitude absence of goblet cell metaplasia in IL-4 KO and IL-10 KO mice of the pulmonary inflammatory response was significantly greater provides additional evidence that these cytokines are crucial for in WT mice (Fig. 5, B, F, and J) than in IL-4 KO (Fig. 5, C, G, and driving the allergic response during C. neoformans infection in K) or IL-10 KO mice (Fig. 5, D, H, and L). The inflammatory C57BL/6 mice. In addition, because pulmonary clearance of the infiltrate in the lungs of WT mice consisted mainly of neutrophils, fungal infection is greater in IL-4 KO and IL-10 KO than WT macrophages and eosinophils and mirrored the quantitative anal- mice, these data also indicate that the type rather than simply the ysis of whole lung enzymatic digests (Figs. 3 and 4). The limited magnitude of the pulmonary inflammatory response is a critical inflammation in IL-4 KO and IL-10 KO mice largely consisted of factor for clearing the infection. mononuclear cells. Slightly enhanced pulmonary fibrosis was evident in sections from WT mice (Fig. 5, J–L). However, the most Characterization of the T1/T2 immune response in the lung and striking histological difference was the lack of goblet cell meta- LALN plasia in IL-4 KO and IL-10 KO mice compared with WT mice To analyze the T1/T2 polarization of the response, IFN-␥, IL-4, (Fig. 5, E–H), which correlated with the decreased pulmonary IL-5, and IL-13 production by lung leukocytes was analyzed at IL-13 levels in IL-4 KO and IL-10 KO mice (Fig. 6). Goblet cell weeks 1 and 2 postinfection. Total lung leukocytes were isolated metaplasia is a significant feature of allergic airway disease and the and cultured. To control for possible differences in the amount of The Journal of Immunology 1031 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

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, ϫ250. cryptococci (Ag) between the different groups, cultures were set up not augmented in IL-10 KO mice. The production of T2 cytokines with either no additional Ag or with 107 HKC (Ͼ20:1 ratio of by lung leukocytes from IL-4 KO and IL-10 KO mice was lower exogenous to endogenous cryptococci). Cytokines were measured than that from WT mice and the addition of exogenous Ag did not in the supernatants after 24 h of culture. Lung leukocytes from completely abrogate the differences. WT, IL-4 KO, and IL-10 KO mice all produced significant levels We next analyzed the draining lymph nodes (LALN) for evi- of IFN-␥ in the presence or absence of additional Ag (Fig. 6). In dence of changes in T1 vs T2 polarization. Despite the presence of contrast, lung leukocyte production of IL-4, IL-5, and IL-13 in the low numbers of cryptococci in the LALN (Fig. 1), these cells did absence of exogenous Ag was significantly lower in both IL-4 KO not produce cytokines in vitro unless restimulated with HKC (Fig. and IL-10 KO mice compared with WT mice (Fig. 6). In the pres- 7). At both weeks 1 and 2, LALN cells from WT mice produced ence of exogenous Ag in the cultures, IL-5 and IL-13 were sig- IFN-␥, IL-5, and IL-13 (Fig. 7). Production of IL-4 was minimal. nificantly lower at week 2 in both IL-4 KO and IL-10 KO mice There was no difference in IFN-␥, IL-4, IL-5, or IL-13 production compared with WT mice (Fig. 6). HKC-stimulated IL-4 levels by LALN cells from WT or IL-10 KO mice (Fig. 7). In contrast to from IL-10 KO lung leukocytes were significantly lower at week LALN cells from IL-10 KO mice, LALN cells from IL-4 KO mice 1 and did not increase at week 2. HKC-stimulated IL-13 levels produced significantly lower levels of IL-13 and IL-4 (Fig. 7) IL-5 were significantly lower at week 1 in the IL-4 KO lung leukocyte production was also lower at week 2. In addition, serum IgE levels cultures. In summary, the production of IFN-␥ or T2 cytokines was were at uninfected levels in IL-4 KO mice but were significantly 1032 IL-4 AND IL-10 IN T2 IMMUNITY DURING ABPM Downloaded from http://www.jimmunol.org/ FIGURE 6. T1- and T2-type cytokine production by lung leukocytes. Total lung leukocytes were obtained 1 and 2 wk postinfection and cultured for 24hat5ϫ 106 cells/ml. Lung leukocytes were cultured in the absence of exogenous stimuli (No Ag) or in the presence of HKC at 1 ϫ 107/ml. Supernatants .p Ͻ 0.05 for WT vs IL-10 KO or vs IL-4 KO; n ϭ 11–12 mice/group/time point for weeks 1 and 2 ,ء .were harvested and cytokine levels measured by ELISA elevated in both WT and IL-10 KO mice (Fig. 8). Thus, IL-4 KO Discussion mice do not appear to develop the systemic (LALN and serum) or These studies are the first to focus on the role of IL-4 and IL-10 in local (lungs) T2 responses characteristic of the allergic broncho- the context of a chronic APBM caused by C. neoformans. Both by guest on October 1, 2021 pulmonary C. neoformans infection. In contrast, the systemic T2 IL-4 KO and IL-10 KO mice have enhanced pulmonary clearance elements of the response remain unaltered in IL-10 KO mice of C. neoformans compared with WT C57BL/6 mice. It has been whereas the T2 response in the lungs fails to develop. demonstrated previously that IL-10 KO mice have increased sur- ␣ We also examined whether TNF- and IL-12 production by vival following i.v. inoculation of C. neoformans (strain 24067) lung leukocytes was altered in IL-4 KO and IL-10 KO mice. At and pulmonary infection with C. neoformans NU-2 (35, 36). In week 2, TNF-␣ levels were higher in lung leukocyte cultures from contrast, there was not a difference in survival between WT and IL-4 KO mice compared with WT mice. But there was no differ- IL-4 KO mice in these studies (35, 37). Interestingly, delayed-type ence in lung leukocyte production of IL-12 or IL-10 between WT hypersensitivity responses were reduced in IL-4 KO mice follow- and IL-4 KO mice (Fig. 9). However, lung leukocyte production of ing infection with the less virulent C. neoformans strain 184A but TNF-␣ and IL-12 was significantly elevated and IL-10 absent at splenic T1 cytokine responses were increased (37). IL-5 and IL-10 weeks 1 and 2 in IL-10 KO mice (Fig. 9). TNF-␣ production was production by splenocytes was down-regulated in IL-4 KO also significantly elevated in LALN leukocytes at week 2 (Fig. 9). C. neoformans Thus, in the absence of IL-10, local (pulmonary) TNF-␣ and IL-12 C57BL/6 mice. However, in i.v. infection with ϫ production by lung leukocytes is up-regulated, favoring the man- 1841 in B6 129F2 mice, there was a significant enhancement of ifestation of a T1 response in the lungs. survival in IL-4 KO mice (20). Similar findings were reported using anti-IL-4 mAb to treat CDF1 mice infected intratracheally Role of IFN-␥ in controlling C. neoformans growth in the lungs with C. neoformans YC-11 (15). To put all these studies in the Because IFN-␥ production by LALN or lung leukocytes did not context of our current results and model, it needs to be noted that significantly change in the absence of IL-4 or IL-10, even though none of the animal model systems just described are true “chronic” clearance was enhanced, this raised the issue of whether IFN-␥ pulmonary infections; in other words, all of the control animals plays a role in controlling the growth of C. neoformans in the lungs still die. In contrast, C57BL/6 mice (6–8 wk at time of infection) of C57BL/6 mice. To address this question, IFN-␥ KO C57BL/6 infected with C. neoformans strain 24067 can survive Ͼ12 wk mice were infected and the pulmonary burden at 3 wk postinfec- while harboring a stable C. neoformans burden on the lungs of 6 7 tion was compared with WT mice. As illustrated in Fig. 10, the 10 –10 CFU (3, 4). Thus, the role of IL-4 is going to be influenced pulmonary burden in WT mice was high but the number of organ- by the cytokine milieu and virulence of the cryptococcal strain. The isms in the lungs of IFN-␥ KO mice at this time point was signif- major findings of our current study are that in ABPM caused by C. icantly higher. Thus, despite production of potentially antagonistic neoformans: 1) IL-4 and IL-10 promote the development of a T2 cytokines during cryptococcal infection in C57BL/6 mice, IFN-␥ response (eosinophilia, IL-4, IL-5, IL-13) in the lungs and the subse- is required to limit the extent of the infection with pulmonary CFU quent chronic pulmonary infection; 2) IFN-␥ induction is independent levels being 50-fold higher in IFN-␥ KO C57BL/6 mice. of the influence of IL-4 and IL-10; and 3) IL-4 promotes T2 type The Journal of Immunology 1033 Downloaded from http://www.jimmunol.org/

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 ϫ 106 cells/ml. Cells were cultured in the absence of exogenous stimuli (No Ag) or in the presence p Ͻ 0.05 for WT vs IL-10 KO or vs IL-4 KO; n ϭ 11–12 ,ء .of HKC at 1 ϫ 107/ml. Supernatants were harvested and cytokine levels measured by ELISA mice/group/time point. anti-cryptococcal immunity systemically and at the site of infection is normally produced during inflammatory responses as one mech- whereas IL-10 only promotes a T2 anti-cryptococcal response locally anism to regulate inflammatory responses and prevent over- by guest on October 1, 2021 (lungs). exuberant inflammation such as is seen in inflammatory bowel C. neoformans-infected C57BL/6 mice produced both T1 and disease (46). IL-12 and TNF-␣ are required for the development of T2 cytokines in their lungs and LALN. Previous studies have protective pulmonary T1 responses to C. neoformans (9, 11, 14, shown that LALN and lung cells from C. neoformans-infected 16, 20–22, 47). Thus, the high levels of IL-12 and TNF-␣ in C. C57BL/6 mice fail to produce IL-2 and secrete very low levels of neoformans-infected IL-10 KO C57BL/6 mice is consistent with IFN-␥ when compared with resistant mice (3). Our studies showed the strong T1 response in these mice. that C57BL/6 lung leukocytes and lymph node cells secrete sig- Another observation from these studies is that strong polariza- nificant levels of IFN-␥. However, despite the secretion of IFN-␥, tion of the cell-mediated response to C. neoformans does not ap- C57BL/6 mice develop T2 responses in their lungs and lymph pear to occur in the lymph nodes. Rather, early polarization of the nodes. Also, previous data had shown that C57BL/6 mice produce response appears to occur largely at the site of infection, the lungs, elevated levels of IL-5 and develop chronic eosinophilia in their despite the presence of cryptococci in the draining lymph nodes. 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-␣. Thus, lung and LALN leukocytes from C57BL/6 mice secrete IFN-␥, IL-12, and TNF-␣ as well as IL-4, IL-5, and IL-13, creating a mixed T1/T2 environment in the lungs and lung draining lymph nodes. How- ever, 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-␣ 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 FIGURE 8. Total serum IgE levels in C. neoformans-infected mice. To- myeloid and lymphoid leukocytes following exposure to C. neo- tal serum IgE levels were determined by ELISA. Dotted line represents IgE p Ͻ 0.05, comparing WT vs IL-10 KO or ,ء .formans, including TNF-␣ and IL-12 (39–43). The polysaccharide levels for uninfected mice capsule of C. neoformans (glucuronoxylomannan) is a potent in- IL-4 KO mice; n ϭ 11–12 mice/group/time point for weeks 1 and 2 and 6 ducer of IL-10 from leukocytes (40, 42, 44, 45). In addition, IL-10 mice/group/time point for week 3. 1034 IL-4 AND IL-10 IN T2 IMMUNITY DURING ABPM Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

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 ϫ 106 cells/ml. Cells were cultured in the absence of exogenous stimuli (No Ag) or in the presence of HKC at 1 ϫ p Ͻ 0.05 for WT vs IL-10 KO or vs IL-4 KO; n ϭ 11–12 mice/group/time ,ء .ml. Supernatants were harvested and cytokine levels measured by ELISA/107 point for weeks 1 and 2 and 6 mice/group/time point for week 3.

For the most part, both T1 and T2 responses coexist in the LALN Surprisingly, despite the higher production of TNF-␣ and IL-12 of C. neoformans-infected C57BL/6 mice, similar to the LALN in the lungs of IL-10 KO mice, and the reduction of T2 cytokines ϫ ␥ from B6 129F2 mice (13). Both IL-4 KO and IL-10 KO in both IL-4 KO and IL-10 KO mice, the levels of IFN- were not C57BL/6 mice have a significantly diminished allergic (T2) re- significantly enhanced in these mice. One possibility is that, in the sponse in the lungs and are able to begin to clear the infection. At absence of IL-4 and IL-10, the levels of IFN-␥ are sufficient to this stage of the investigation, we are not sure of the cellular drive protective immunity and other non-IL-4 or non-IL-10 mech- sources of the cytokines in the LALN vs lungs or whether they are anisms keep an overexuberant IFN-␥ response from developing. In different in IL-4 KO vs IL-10 KO vs WT. However, these studies other studies, it appears that a non-T cell source of IFN-␥ is re- demonstrate that the polarization of the cellular response in the sponsible for the low-level protection of C57BL/6 during pulmo- LALN vs lungs is different. Is it possible that T cells polarize in the nary cryptococcosis.5 The results from the current studies indicate LALN and then immediately emigrate to the lungs? We cannot that the production of IFN-␥ is independent of regulation by IL-4 formally exclude this possibility in this model, but in other studies and IL-10. Furthermore, IFN-␥ is required for control of the in- from our laboratory that have addressed this question during pro- fection. In studies of allergic bronchopulmonary cryptococcosis tective (T1) immunity to C. neoformans, it is clear that CD4 T cell using IFN-␥ KO C57BL/6 mice, it appears that IFN-␥ production polarization does not occur in the LALN, blood or spleen but is is not required for the development of T2 cytokine producing cells evident in the lungs.4 Our studies indicate that IL-4 also promotes in the lungs and LALN and T2 cytokine (IL-4, IL-5, IL-13) pro- the development of the T2 response in the LALN whereas IL-10 duction was enhanced at a number of time points in IFN-␥ KO does not play any role in regulating the response in the LALN compared with WT C57BL/6 mice.5 during an allergic response to C. neoformans.

5 S. A. Arora, Y. Hernandez, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, 4 D. M. Lindell, T. A. Moore, R. A. McDonald, G. B. Toews, and G. B. Huffnagle, and G. B. Huffnagle, Role of IFN-␥ in regulating T2 immunity and the development Polarization of CD4ϩ T cells occurs in non-lymphoid tissues. Submitted for of alternatively activated macrophages during allergic bronchopulmonary mycosis. publication. Submitted for publication. The Journal of Immunology 1035

flammatory 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 CD4ϩ 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 (56–58). Regulatory T1 T cells are FIGURE 10. Pulmonary levels of C. neoformans in WT and IFN-␥ KO mice at week 3 postinfection following intratracheal inoculation (104 one of the subsets of regulatory T cells that express the markers low -p Ͻ 0.05, comparing WT vs IFN-␥ KO mice; n ϭ 6 mice/group. CCR5, T1-ST2, CD25, CD45RO, and CD45RB . Most regula ,ء .(CFU tory T1 T cells exclusively secrete high levels of IL-10, which they use as the suppressor mechanism, but some can also secrete IL-5 Although the in vivo anti-inflammatory properties of IL-10 are and TGF-␤. They produce little or no IL-2, thereby proliferating fairly consistent throughout the literature, our studies demonstrate poorly, and produce no IL-4 or IFN-␥. Studies performed in the Downloaded from that the fungal agent may be an important consideration in under- pulmonary pathogen Bordetella pertussis, have shown that regu- standing the role of IL-10 in ABPM. Pulmonary cryptococcosis latory T1 T cells can prevent the development of Th1 responses with strain 24067 in C57BL/6 mice shares many features with through the production of IL-10, introducing a new strategy used murine models of allergic bronchopulmonary aspergillosis. These by pathogens in the respiratory tract to prolong survival of a patho- include high IgE, elevated peripheral blood and lung eosinophils, gen (56). Such a mechanism may occur in allergic bronchopulmo- pulmonary inflammation, elevated levels of IL-4, IL-5, and IL-13, nary cryptococcosis; however, IL-10 seems to be playing a posi- http://www.jimmunol.org/ production of IFN-␥, pulmonary fibrosis, goblet cell hyperplasia tive role in the pulmonary T2 response rather than a negative role and chronic fungal colonization/persistence (3, 4, 48–51). In mu- in the T1 response because production of IFN-␥ by lung leuko- rine models of allergic bronchopulmonary aspergillosis, the T2 cytes appears to be unaltered in IL-10 KO mice (however, the cytokines IL-4, IL-5, and IL-13 are required for these pathologic cellular source of IFN-␥ may be different in WT vs IL-10 KO features of the host response (48–51). Both IL-4 and IL-10 can mice). 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 As- References pergillus Ag-induced inflammation, the allergic lung response is 1. Lovchik, J. A., J. A. Wilder, G. B. Huffnagle, R. Riblet, C. R. Lyons, and by guest on October 1, 2021 similar in intranasally primed IL-10 KO and WT C57BL/6 mice M. F. Lipscomb. 1999. Ig heavy chain complex-linked genes influence the immune response in a murine cryptococcal infection. J. Immunol. 163:3907. (52). However, pulmonary eosinophilia is heightened in IL-10 KO 2. Huffnagle, G. B., G. H. 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