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The Journal of Immunology, 2007, 178: 1477-1487.
Copyright © 2007 by The American Association of Immunologists, Inc.

Targeting of CD25 and Glucocorticoid-Induced TNF Receptor Family-Related Gene-Expressing T Cells Differentially Modulates Asthma Risk in Offspring of Asthmatic and Normal Mother Mice1

Cedric Hubeau2,*, Irina Apostolou{dagger} and Lester Kobzik3,*,{ddagger}

* Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115; {dagger} Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115; and {ddagger} Department of Pathology, Brigham & Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological mechanisms leading to increased asthma susceptibility in early life remain obscure. In this study, we examined the effects of neonatal Ab treatments targeting T cell populations on the development of an asthma syndrome. We used a model of increased asthma susceptibility where offspring of asthmatic BALB/c mother mice are more prone (than normal pups) to develop the disease. Neonatal pretreatment of naive pups with mAb directed against the IL-2R{alpha} chain (CD25), the costimulatory molecule glucocorticoid-induced TNFR family related gene, and the inhibitory molecule CTLA-4 elicited contrasting effects in offspring depending on the mother’s asthma status. Specifically, neonatal CD25high T cell depletion stimulated asthma susceptibility in normal offspring whereas it ameliorated the condition of pups born of asthmatic mothers. Conversely, glucocorticoid-induced TNFR family related gene ligation as a primary signal reduced the spleen cellularity and largely abrogated asthma susceptibility in asthma-prone offspring, without inducing disease in normal pups. Striking changes in Th1/Th2 cytokine levels, especially IL-4, followed mAb pretreatment and were consistent with the impact on asthma susceptibility. These results point to major differences in neonatal T cell population and responsiveness related to maternal asthma history. Interventions that temporarily remove and/or inactivate specific T cell subsets may therefore prove useful to attenuate early life asthma susceptibility and prevent the development of Th2-driven allergic airway disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The increasing prevalence of asthma is a major issue of public health in industrialized countries. Asthma affects ~10% of the U.S. population and is considered a leading cause of young children hospitalization (1). Allergic asthma has origins in early life (2, 3) and is commonly characterized by airway hyperresponsiveness (AHR)4 to bronchoconstrictors, reversible airflow obstruction, and allergic airway inflammation with eosinophilia (4). Although the exact interrelation between AHR and allergic inflammation is still debated (5, 6), there exists evidence that two major Th2 cytokines, IL-4 and IL-13, are critically involved in most aspects of allergic asthma (7, 8). It is therefore reasonable to speculate that, in asthma-susceptible individuals, IL-4 and/or IL-13 overproduction arises because natural controls are lacking or defective (9).

Naturally occurring CD4+CD25highFoxp3+ T cells (regulatory T cells (Treg)) constitute a subset of T lymphocytes typically involved in immune homeostasis (10). Peripheral Treg are thought to expand during fetal development in humans (11) while they are found after day 3 of life in mice (12). Because Treg appear early in life and are extremely efficacious at restraining inflammatory responses, it has been suggested that Treg also participate in the control of allergic asthma by maintaining the tolerance to nonself Ags (13, 14). In asthmatic patients, however, it is not clear whether Th2-skewed responses are favored by dysfunctional Treg or whether allergic inflammation overwhelms normally functioning Treg (15, 16, 17). To our knowledge, the mechanisms controlling the development of allergic airway disease in newborns have not yet been addressed.

We previously described a murine model of maternal asthma where intentionally suboptimal sensitization and exposure to allergens allow the identification of neonates with increased susceptibility to asthma (18). This model of natural predisposition to asthma has proven useful to investigate immunobiological mechanisms involved in the maternal transfer of asthma risk without other confounding genetic and environmental factors (19, 20, 21). In this study, we have used a variation of the original maternal asthma model by pretreating 4-day-old mouse pups with mAb raised against various Treg markers. Indeed, despite the fact that there exist no "perfect" Treg surface markers, Treg are theoretically the only cells to express high levels of CD25, CTLA-4, and glucocorticoid-induced TNFR family related gene (GITR) in steady state conditions (14), as in naive mouse pups. We have examined the ensuing development of asthma in mAb treated pups born of normal or asthmatic mother mice.

Despite normal Treg frequency and profile, pups born of asthmatic mothers showed increased splenic cellularity (including CD4+CD25–Foxp3– T cells) before allergen encounter. Although only asthma-prone offspring developed Th2 cytokine imbalance, allergic airway inflammation, and AHR in response to the suboptimal asthma protocol, in vivo mAb pretreatment interfering with T cell/Treg populations before allergen encounter greatly ameliorated asthma susceptibility in these pups born of asthmatic mothers.


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

Four-day-old BALB/c litters and 8- to 10-wk-old adult males were obtained from Charles River Laboratories. The mice were housed in a pathogen-free barrier facility. All animal experiments were conducted under a protocol approved by our institutional review board.

Allergen sensitization and aerosol exposure

The model of maternal asthma using OVA-sensitized and aerosol-exposed BALB/c female mice was detailed previously (18). The modification of this protocol is represented in Fig. 1. Briefly, future asthmatic mothers were sensitized by two i.p. injections of 5 µg of OVA (grade IV; Sigma-Aldrich) emulsified in 1 mg of aluminum hydroxide (J. T. Baker) on days 5 and 9 of life. At 3 wk of age, the females were placed in separate cages and repeatedly exposed to 3% OVA aerosols (10 min.) every 4 wk for 3 mo, until mating with normal BALB/c males. Naive offspring born of normal (control) and asthmatic mother mice received mAb on day 4 of life (see below) and were submitted to an intentionally suboptimal protocol of asthma induction that features a single (rather than two) i.p. injection of 5 µg of OVA in 1 mg of aluminum hydroxide (day 5) followed by 3% OVA aerosols (days 12–14). Final physiologic and pathologic analyses were performed on days 15 and 16, as described previously (18).


Figure 1
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  		FIGURE 1. Model of maternal asthma and suboptimal asthma induction in naive pups. Future asthmatic mother mice were fully OVA-sensitized as pups and repeatedly exposed to OVA aerosols every 4 wk. Their newborn offspring were submitted to an intentionally suboptimal protocol of asthma induction (day 5 and days 12–14). When indicated, 4-day-old naive pups were pretreated with mAb.

 
In vivo mAb treatment of naive pups

Low endotoxin, azide-free, mAb raised against mouse CD25 (rat IgG1, clone PC61) and mouse GITR (rat IgG2b, clone DTA-1) were purchased from BioExpress Cell Culture Services. Anti-mouse CTLA-4 (hamster IgG1, clone UC10-4F10) was a gift from Dr. P. Finn (Brigham and Women’s Hospital, Boston, MA). Offspring were i.p. injected with 100 µg of mAb on day 4 of life, 24 h before starting the suboptimal asthma protocol (day 5 and on, see above and Fig. 1). These experiments included pups injected with 100 µg of purified control IgG (Sigma-Aldrich) and referred to as "normal" or "asthma" (i.e., born of asthmatic mothers) in the figures. Experiments focusing on GITR ligation as a primary vs costimulatory signal also included pups injected with DTA-1 mAb 24 h after OVA sensitization and referred to as "After OVA".

Collection of splenic cells from 7-day-old pups and assessment of spleen cellularity

Naive pups were injected with 100 µg of mAb or purified control IgG on day 4 of life (see above). To delineate the effects of mAb treatments without confounding immune responses due to asthma induction, these pups were not OVA sensitized. The spleens were collected on day 7 of life and placed in chilled HBSS (BioWhittaker). Splenocytes were then expelled from the splenic capsule through a 70-µm nylon mesh filter. After RBC lysis, living cells were counted by means of a hemocytometer and trypan blue staining (Sigma-Aldrich).

Flow cytometric analysis

Eight million splenocytes were dispensed in each 1.5-ml microfuge tube. After incubation with PBS containing 5% rat serum and 10% rat anti-mouse CD16/32 (Fc block, clone 93; eBioscience), a mixture of mAb was added to each tube. This step included Pacific Blue-conjugated rat anti-mouse CD4 (clone L3T4; BD Biosciences), biotinylated rat anti-mouse CD25 (clone 7D4; BD Biosciences), and allophycocyanin-conjugated rat anti-mouse GITR (clone DTA-1, eBioscience). Then the cells were washed with PBS and a second labeling was performed, including 7-aminoactinomycin D (7-AAD; BD Biosciences) and allophycocyanin-Cy7-conjugated streptavidin (BD Biosciences). To prevent the diffusion of 7-AAD from dead cells all the buffers thereafter contained actinomycin D (1/2000). After washing with PBS and incubation with a permeabilization buffer containing 0.1% saponin and 0.009% sodium azide (eBioscience), the cells were incubated again with 5% rat serum and 10% Fc block before adding FITC-conjugated rat anti-mouse Foxp3 (clone FJK-16s; eBioscience) and PE-conjugated hamster anti-mouse CTLA-4 (clone UC10-4F10; BD Biosciences). The cells were finally washed twice with the permeabilization buffer and resuspended in a fixation buffer containing paraformaldehyde (eBioscience). Six-color flow cytometric analysis was performed with a FACSAria (BD Immunocytometry Systems). Lymphocytes were gated according to forward and side light-scatter properties, then dead cells were excluded according to 7-AAD staining. Nonlabeled cells and cells incubated with control isotypes were used to determine the background noise. Cells labeled with a single fluorochrome were used to adjust the separation thresholds of the cytometer channels. Acquisition was set for 4 million events and final analysis was performed on a semilogarithmic scale using FlowJo for Macintosh (version 6.0, Tree Star).

Pulmonary function testing and pathologic analysis

Airway responsiveness to increasing doses of aerosolized methacholine chloride (MCh; Sigma-Aldrich) was evaluated in 2-wk-old pups, 24 h after last allergen aerosol challenge. Unrestrained plethysmography, in particular the use of enhanced pause (Penh), as a surrogate of the mouse lung function is a common, yet controversial technique (22). It is worth noting, however, that more invasive techniques are not currently available for very small animals such as 2-wk-old pups weighing <10 g. In addition, Penh and compliance were demonstrated to both highly correlate with lung resistance in the BALB/c mouse strain used exclusively in our studies (23). Bronchoalveolar lavage (BAL) was performed on euthanized animals 48 h after last allergen aerosol challenge. Total BAL cellularity was evaluated by means of a hemocytometer and cells stained with Türk’s solution (Merck), while differential counts were performed on cytocentrifuge slides stained with Diff-Quick (VWR).

Multiplex cytokine assay

Cytokines were assayed in serum and BAL samples using xMAP technology (Luminex) and multiplex immunoassay kits purchased from Linco Research. Color-coded microspheres were set to detect mouse IFN-{gamma}, IL-4, IL-10, IL-13, and either IL-5 (in BAL samples) or IL-12p70 (in serum samples). Analysis was performed using Masterplex QT 2.0 software. Graphs are representative of data averaged from three to six individual samples assayed in duplicate. Detection limits for each cytokine ranged from 0.3 pg/ml (IL-4) to 10.3 pg/ml (IL-10). Concentrations plotted as zero on the graphs were below detection limits.

Statistical analysis

Data are presented as mean ± SEM. Differences between groups were compared using a one-way ANOVA adjusted for multiple comparisons with protected least significant differences Fisher’s test or Student’s t test when appropriate. StatView software program (Abacus Concepts) was used for statistical analysis and statistical significance was accepted for p values <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Naive asthma-prone offspring show increased spleen cellularity and naive T cell numbers despite phenotypically normal Treg population

Because we sought to investigate the role of Treg in early asthma susceptibility, we targeted these cells in newborn mice with or without increased asthma susceptibility. Two types of in vivo treatments were performed in 4-day-old naive pups, either aimed to deplete CD25high T cells by means of PC61 mAb (14, 24, 25), or to abrogate Treg function by means of DTA-1 mAb agonist of the costimulatory molecule GITR (26, 27). Spleen cells were collected, counted, and analyzed by flow cytometry 3 days later.

First, spleen cellularity was assessed to evaluate the effects of mAb treatments at the systemic level. An intrinsic difference in the pups’ spleen cellularity was found depending on the mother’s asthma status (Fig. 2). Naive offspring born of asthmatic mothers showed 2-fold higher splenocyte numbers compared with normal pups (p = 0.0036). Although PC61 treatment appeared not to affect this intrinsic difference, DTA-1 treatment led to opposing effects in pups, also depending on the mother’s asthma status. Specifically, DTA-1 treatment led to increased spleen cellularity (p = 0.0468) in normal pups whereas it led to decreased spleen cellularity (down to normal levels) in pups born of asthmatic mothers (p = 0.0008).


Figure 2
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  		FIGURE 2. Spleen cellularity in 7-day-old pups. Spleen cellularity was assessed in 7-day-old pups that were treated (or not) with mAb against CD25 (PC61) or GITR (DTA-1). Naive asthma-prone pups (referred to as asthma) showed increased spleen cellularity compared with normal pups (p = 0.0036). This intrinsic difference was unchanged in pups that had received PC61 mAb on day 4 of life (compared with untreated pups). In sharp contrast, DTA-1 pretreatment (day 4) led to increased spleen cellularity in normal pups (p = 0.0468) and decreased spleen cellularity in asthma-prone offspring (p = 0.0008).

 
To verify the effects of mAb treatments on CD4+ splenic T cell population, flow cytometric analysis was performed and included the markers CD25 and Foxp3 that together allow the discrimination of Treg from other T cells. As expected, Treg represented ~5–7% of peripheral CD4+ T cells in pups, regardless of the mother’s asthma status (Fig. 3A). Specifically, normal Foxp3+CD25+ Treg frequency was 6.86 ± 1.34 vs 6.22 ± 1.95 in asthma-prone pups (Fig. 3A and data not shown). As previously reported (14, 24, 25), PC61 mAb depleted CD25high Treg with little effect on other T cell subsets, including CD25dim T cells (Fig. 3B). DTA-1 mAb treatment appeared not to modulate splenic CD4+ T cell ratios (Fig. 3C).


Figure 3
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  		FIGURE 3. Splenic CD4+ T cell phenotype and effects of mAb on T cell subsets. A, Flow cytometric analysis of splenic CD4+ lymphocytes showed that frequencies of different T cells subsets were generally similar between normal and asthma-prone pups. B, Pretreatment with PC61 mAb (day 4) showed effective depletion of CD4+CD25highFoxp3+ Treg in 7-day-old pups. C, Pretreatment with DTA-1 mAb (day 4) had no apparent effect on the frequencies of different T cells subsets, as assessed in 7-day-old pups. D, The frequencies of the different splenic CD4+ T cell subsets expressing CTLA-4 and GITR were also comparable in 7-day-old normal and asthma-prone pups. Of note, CD4+CD25highFoxp3+ Treg constituted the large majority of splenic CD4+ T cells expressing both CTLA-4 and GITR (absolute numbers of GITR+CTLA-4+ cells are indicated on the top right of each panel). E, Cell counts of different CD4+ T cell subpopulations showed that Foxp3–CD25– T cells constituted the large majority of splenic CD4+ T cells and were more numerous in asthma-prone offspring compared with their normal counterparts (p = 0.0034). Very few Foxp3–CD25+ effector T cells were found in these pups, regardless of the mother’s asthma status or mAb pretreatment. Foxp3–CD25– naive T cell numbers were subjectively increased in DTA-1 pretreated normal pups (not statistically significant, p = 0.057), whereas DTA-1 pretreatment led to decreased Foxp3–CD25– T cell numbers in asthma-prone pups (p = 0.0007).

 
Further characterization of peripheral Treg and non-Treg subpopulations included CTLA-4 and GITR expression (Fig. 3D). Splenic CD4+ T cells collected from normal and asthma-prone pups displayed comparable profiles in terms of frequencies and absolute numbers of CTLA-4 and GITR-expressing cells. Importantly, Treg represented the large majority of T cells expressing high levels of both molecules in the spleen of 7-day-old naive pups, regardless of the mother’s asthma status.

Despite phenotypically similar CD4+ T cell subpopulations, the higher spleen cellularity found in asthma-prone pups corresponded to increased CD4+ T cell numbers (Fig. 3E). Notably, CD25–Foxp3– T cells (representing the large majority of splenic CD4+ T cells in 7-day-old pups) were substantially more numerous in asthma-prone pups compared with their normal counterparts (p = 0.0034). Also, DTA-1 treatment of asthma-prone pups led to a significant decrease (down to normal levels) in CD25–Foxp3– naive T cell numbers (p = 0.0007), whereas a trend toward increase in DTA-1 pretreated normal pups was not statistically significant, (p = 0.057).

These results thus indicate that naive pups born of asthmatic mothers have an intrinsically higher spleen cellularity, in part due to increased CD4+CD25–Foxp3– T cell population. DTA-1 treatment modulated the spleen cellularity and CD4+ T cells numbers in offspring, depending on the mother’s asthma status. Additionally, PC61 mAb was confirmed to effectively deplete CD25high Treg in vivo. We next sought to determine the effects of these mAb interventions on asthma susceptibility.

PC 61 pretreatment increases or reduces asthma susceptibility in pups depending on the mother’s asthma status

Having confirmed that PC61 treatment of 4-day-old newborn mice mainly targets CD25high Treg in vivo, we examined the effects of Treg depletion on normal and asthma-prone baby mice submitted to the suboptimal asthma protocol and tested at 2 wk of age.

Consistent with the original model (18), histopathological examination showed that pups born of asthmatic mothers, when submitted to suboptimal asthma induction, develop substantial inflammatory infiltration in their airways, comprised of eosinophils and mononuclear cells surrounding the bronchi and vessels (Fig. 4A). PC61 pretreatment did not modulate this pattern (Fig. 4B). In contrast, normal pups do not develop allergic airway inflammation following the suboptimal asthma protocol (Fig. 4C). However, PC61 pretreated normal pups, when submitted to the suboptimal asthma protocol, showed an inflammatory infiltration of their airways by eosinophils and mononuclear cells (Fig. 4D).


Figure 4
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  		FIGURE 4. Effects of anti-CD25 (PC61) pretreatment on asthma susceptibility. All offspring were submitted to the suboptimal asthma protocol reported in Fig. 1. Histopathological examination of the airways of (A) untreated vs (B) PC61 pretreated asthma-prone offspring, and (C) untreated vs (D) PC61 pretreated normal pups. An inflammatory infiltrate comprised of mononuclear cells and eosinophils was found in PC61 pretreated but not in untreated normal pups. In BAL samples (E), cellularity was increased in PC61 pretreated normal pups (p < 0.0001) but decreased in PC61 pretreated asthma-prone pups (p = 0.0017). F, BAL differential counts showed that PC61 pretreated normal pups developed eosinophilia (p < 0.0001) and increased percentage of lymphocytes (#, p < 0.0001). During an aerosol challenge with increasing doses of MCh (G), Penh values obtained from PC61 pretreated normal pups reached levels usually found in asthma-prone untreated offspring. PC61 pretreated asthma-prone pups showed Penh values not statistically different from those found in untreated normal pups (***, p < 0.0001, compared with untreated normal pups) (n = 30–75 pups/group).

 
BAL sample analysis confirmed these results and showed that suboptimal asthma induction in PC61 pretreated normal pups increased inflammatory cell numbers (Fig. 4E, p < 0.0001) along with higher eosinophil and lymphocyte percentages (Fig. 4F, p < 0.0001 for both) compared with untreated normal pups. In sharp contrast, PC61 pretreatment of asthma-prone pups appeared to decrease their susceptibility to suboptimal asthma induction, as demonstrated by significantly reduced cell numbers in BAL samples (Fig. 4E, p = 0.0017), albeit with unchanged eosinophil and lymphocyte percentages compared with untreated asthma-prone offspring (Fig. 4F).

When submitted to a MCh aerosol challenge, PC61 pretreated normal pups showed increased Penh values (Fig. 4G, p < 0.0001 for all MCh doses, PC61 treated vs untreated normal pups). In contrast, but consistent with the above data on BAL analysis, PC61 pretreatment led to lower Penh values in asthma-prone pups submitted to suboptimal asthma induction (no statistically significant differences between untreated normal pups and PC61 pretreated asthma-prone pups).

Thus, CD25high Treg depletion before suboptimal asthma induction elicited complex and contrasting effects in offspring, depending on the mother’s asthma status. Although normal pups were rendered highly susceptible to asthma, offspring born of asthmatic mothers developed a substantially milder asthma syndrome.

UC10-4F10 pretreatment stimulates asthma susceptibility in normal pups with minimal benefits for offspring born of asthmatic mothers

Treg suppressive function may depend on CTLA-4 expression (28, 29, 30, 31). Because PC61 pretreatment unexpectedly ameliorated asthma susceptibility in offspring born of asthmatic mothers, we investigated whether CTLA-4 blockade could also have beneficial effects against early asthma susceptibility. Similar to the protocol reported above, we injected anti-mouse CTLA-4 mAb (clone UC10-4F10) into 4-day-old normal and asthma-prone pups before submitting them to the suboptimal asthma protocol.

Histopathological examination of lung tissues revealed a mild inflammatory cell infiltration (eosinophils and mononuclear cells) around the airways and vessels of normal as well as asthma-prone pups pretreated with UC10-4F10 mAb (Fig. 5, A and B, respectively). Analysis of BAL samples confirmed these results, with UC10-4F10 pretreated normal pups having increased inflammatory cell numbers (Fig. 5C, p = 0.0071) and eosinophilia (Fig. 5D, p = 0.0002) compared with untreated normal pups. UC10-4F10 pretreatment did not significantly modulate inflammatory cell numbers in the airways of asthma-prone pups, though it reduced lymphocyte percentages (Fig. 5D, p = 0.005). During a MCh aerosol challenge UC10-4F10 pretreated normal pups showed increased Penh values compared with their untreated counterparts (Fig. 5E, p = 0.0048 at 50 mg/ml and p < 0.0001 at 100 mg/ml). No substantial effect (e.g., increased or decreased Penh values) was found in UC10-4F10 pretreated offspring born of asthmatic mothers.


Figure 5
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  		FIGURE 5. Effects of anti-CTLA-4 (UC10-4F10) pretreatment on asthma susceptibility. All offspring were submitted to the suboptimal asthma protocol reported in Fig. 1. Histopathological examination revealed similarly mild inflammatory infiltrates in the airways of UC10-4F10 pretreated normal (A) vs asthma-prone offspring (B). In BAL samples, cellularity (C) and eosinophilia (D) were increased (p = 0.0071 and p = 0.0002, respectively) in UC10-4F10 pretreated normal pups whereas the percentage of lymphocytes was decreased (#, p = 0.005) in UC10-4F10 pretreated asthma-prone offspring. During an aerosol challenge with increasing doses of MCh (E), Penh values obtained from UC10-4F10 pretreated pups were increased to levels usually found in untreated asthma-prone offspring (**, p = 0.0048, and ***, p < 0.0001, compared with untreated normal pups) (n = 10–12 pups/group).

 
These experiments therefore confirmed that CTLA-4 is critical for preventing normal pups from developing allergic airway disease. Compared with anti-CD25 mAb (PC61), anti-CTLA-4 mAb (UC10-4F10) pretreatment showed minimal beneficial effects on the asthma susceptibility of offspring born of asthmatic mothers.

DTA-1 pretreatment attenuates asthma susceptibility

GITR ligation alone, not as a costimulatory signal, has little effect on T cells or inflammation (32, 33). The fact that GITR-deficient T cells proliferate more than their wild-type counterparts (34), however, suggests that GITR signal, under certain conditions, can also regulate T cell population (35). In this study, we sought to evaluate the effect of GITR ligation alone, as a primary signal, on neonatal asthma susceptibility.

Histopathological examination of lung sections showed airways of DTA-1 pretreated normal pups free of inflammation (Fig. 6A). Interestingly, the airways of DTA-1 pretreated asthma-prone offspring also showed little evidence of inflammation (Fig. 6B). Analysis of BAL samples confirmed these results, with unchanged inflammatory cell counts (Fig. 6C) and differential counts (Fig. 6D) in DTA-1 pretreated normal pups submitted to the suboptimal asthma protocol. Conversely, DTA-1 pretreated asthma-prone pups showed significantly reduced inflammatory cell counts (Fig. 6C, p = 0.0077), and reduced eosinophilia (Fig. 6D, p = 0.006) in BAL samples. During a MCh aerosol challenge and compared with untreated asthma-prone pups (i.e., positive controls), the Penh values obtained from DTA-1 pretreated pups (including normal and asthma-prone offspring) were all similar to those of untreated normal pups (Fig. 6E, p < 0.0001 for all MCh doses, compared with untreated asthma-prone pups).


Figure 6
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  		FIGURE 6. Effects of anti-GITR (DTA-1) pretreatment on asthma susceptibility. All offspring were submitted to the suboptimal asthma protocol reported in Fig. 1. Histopathological examination of the airways of DTA-1 pretreated normal (A) vs asthma-prone offspring (B) showed minor signs of inflammatory infiltration in either group. BAL samples analysis showed reduced cellularity (C) and reduced eosinophilia (D) (p = 0.0077 and p = 0.006, respectively) in DTA-1 pretreated asthma-prone pups compared with their untreated counterparts. DTA-1 pretreated normal pups were indistinguishable from untreated normal pups. During an aerosol challenge with increasing doses of MCh (E), Penh values obtained from DTA-1 pretreated pups remained at levels usually found in untreated normal pups (***, p < 0.0001, untreated asthma-prone pups compared with untreated normal offspring) (n = 16–75 pups/group).

 
GITR ligation alone, before suboptimal asthma induction thus significantly diminished asthma susceptibility in offspring born of asthmatic mothers. Moreover, while beneficial to asthma-prone pups, GITR ligation as a primary signal did not interfere with the natural asthma resistance seen in normal offspring.

DTA-1 treatment after allergen sensitization enhances asthma syndrome

As a costimulatory signal GITR ligation (with GITR-L or DTA-1 mAb) is known to enhance T cell activation induced by TCR/CD3 cross-linking (27, 36). Also, GITR costimulation reportedly breaks Treg suppression in vitro and in vivo (26). Given the results reported above, it was important to differentiate the effects of GITR ligation in naive vs allergen-sensitized pups. We thus tested the prediction that, as reported for adult mice (37), DTA-1 treatment after administration of sensitizing allergen would exacerbate rather than abrogate the disease in normal and asthma-prone pups.

Histopathological examination of lung sections showed that the airways of DTA-1 after OVA-treated normal pups were infiltrated by few inflammatory cells (Fig. 7A). The inflammatory infiltrate was more pronounced in the airways of DTA-1 after OVA asthma-prone offspring (Fig. 7B), albeit without a noticeable effect of DTA-1 mAb treatment per se (see Fig. 4A). Despite these minimal effects on airway histopathology, DTA-1 after OVA treatment led to increased inflammation in BAL samples. In normal pups, this trend did not reach statistical significance in terms of total BAL cellularity (Fig. 7C, p = 0.08), whereas eosinophilia was markedly higher (Fig. 7D, p = 0.0055). In offspring born of asthmatic mothers, DTA-1 after OVA led to increased total cellularity in BAL samples (Fig. 7C, p = 0.0228) but it did not aggravate eosinophilia per se (Fig. 7D). Fig. 7E shows that DTA-1 after OVA also increased Penh values obtained from normal pups (p = 0.02, p = 0.0098, and p = 0.0038 for MCh doses of 25, 50, and 100 mg/ml, respectively, compared with untreated normal pups), while it had little effect on already asthma-prone offspring (who displayed Penh values similar to those from untreated asthma-prone pups).


Figure 7
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  		FIGURE 7. Effects of anti-GITR (DTA-1) treatment after allergen sensitization on asthma susceptibility. DTA-1 mAb were injected after OVA sensitization (day 5), as per the suboptimal asthma protocol reported in Fig. 1. Histopathological examination of the airways of DTA-1 treated normal (A) vs asthma-prone offspring (B) showed inflammatory infiltrates only in the latter group. BAL samples showed increased cellularity in asthma-prone offspring (C) (p = 0.0228) and eosinophilia in normal pups (p = 0.0055) (D). During an aerosol challenge with increasing doses of MCh (E), Penh values obtained from all the pups treated with DTA-1 after OVA sensitization reached levels usually found in untreated asthma-prone offspring (*, p = 0.02, *, p = 0.0098, and ** p = 0.0038 for MCh doses of 25, 50, and 100 mg/ml, respectively, compared with untreated normal pups) (n = 12–15 pups/group).

 
Thus, as reported for adult mice (37), DTA-1 treatment of normal pups, in the course of asthma, enhanced the development of AHR and allergic airway inflammation. In offspring born of asthmatic mothers DTA-1 treatment after suboptimal asthma induction only slightly enhanced allergic airway inflammation.

mAb pretreatments modulate compartmentalized cytokine production in response to suboptimal asthma induction

As reported above, mAb pretreatments differentially influenced the offspring susceptibility to asthma. Given the critical role of cytokines in the orchestration of allergic airway disease (8), we examined the cytokines produced in response to suboptimal asthma induction both locally (BAL) and systemically (serum), in 2-wk-old offspring pretreated (day 4) with IgG, PC61, or DTA-1 mAb.

Fig. 8A shows that the lungs of PC61 pretreated normal pups submitted to the suboptimal asthma protocol produced more IL-4 (p < 0.0001) and IL-13 (p = 0.005), along with reduced IL-10 secretion (p = 0.0204), as compared with their untreated counterparts. Conversely, DTA-1 pretreatment did not appear to modulate the cytokine pattern found in untreated normal pups. In contrast to normal pups, PC61 pretreated asthma-prone pups submitted to the suboptimal asthma protocol produced less IL-13 in their lungs compared with their untreated counterparts (Fig. 8B, p = 0.0107). DTA-1 pretreatment appeared again not to significantly impact cytokine production patterns at the time of the suboptimal asthma protocol.


Figure 8
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  		FIGURE 8. Multiplex cytokine assay of BAL samples. A, Analysis of cytokine production patterns in BAL samples obtained from normal pups submitted to the suboptimal asthma protocol. The airways of PC61 pretreated pups released increased levels of IL-4 (p < 0.0001) and IL-13 (p = 0.005) along with decreased levels of IL-10 (p = 0.0204) in response to suboptimal asthma induction. B, Analysis of cytokine production patterns in BAL samples obtained from asthma-prone offspring submitted to the suboptimal asthma protocol. The airways of PC61 pretreated pups released decreased levels of IL-4 (p = 0.0107, compared with untreated asthma-prone pups) in response to suboptimal asthma induction. Graphs represent data averaged from three to six individual samples assayed in duplicates (see Materials and Methods).

 
At the systemic level (Fig. 9A) PC61 pretreated normal pups submitted to the suboptimal asthma protocol produced increased levels of both IFN-{gamma} (p = 0.0041) and IL-4 (p < 0.0001). This finding contrasting with the data from BAL samples underlines a compartmentalized cytokine production. In this study too, DTA-1 pretreatment did not appear to modulate the cytokine pattern seen in untreated normal pups. In sharp contrast, DTA-1 as well as PC61 pretreated asthma-prone pups submitted to the suboptimal asthma protocol showed significantly reduced IL-4 serum levels compared with untreated asthma-prone offspring (p = 0.006 and p = 0.0031, respectively).


Figure 9
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  		FIGURE 9. Multiplex cytokine assay of serum samples. A, Analysis of cytokine production patterns in serum samples obtained from normal pups submitted to the suboptimal asthma protocol. PC61 pretreated pups showed increased circulating levels of both IFN-{gamma} (p = 0.0041) and IL-4 (p < 0.0001) in response to suboptimal asthma induction. B, Analysis of cytokine production patterns in serum samples obtained from asthma-prone offspring submitted to the suboptimal asthma protocol. Both PC61 and DTA-1 pretreated pups showed decreased circulating levels of IL-4 (p = 0.006 and p = 0.0031, respectively) in response to suboptimal asthma induction. Graphs represent data averaged from three to six individual samples assayed in duplicates (see Materials and Methods).

 
Analysis of BAL and serum samples revealed that mAb pretreatments (day 4) influenced both the local and systemic cytokine production in 2-wk-old pups submitted to the suboptimal asthma protocol. Of important note, IL-4 was the only cytokine to be modulated simultaneously in BAL and serum samples, showing the most concordance with the development of an asthma phenotype.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The model presented here focuses on an intentionally suboptimal protocol of allergen sensitization and exposure that promotes the development of AHR and allergic airway inflammation in asthma-prone offspring but fails to do so in normal pups. This protocol allows the distinction between inbred mice that are differentially susceptible to asthma, in an allergen nonspecific manner (18). This model has facilitated studies on the impact of maternal asthma on exposure to CpGs (20), breastfeeding (21), and allergen-specific T cell function during pregnancy (19). We found this model also suitable to study the role of early T cell subpopulations in the neonatal susceptibility to asthma.

CD4+CD25highFoxp3+ Treg are potent suppressor of inflammatory processes leading to autoimmunity (38), allograft rejection (39), tumor growth (32), and chronic infection to parasites (40). With regards to Treg in allergic disorders, compelling evidence has been provided by Foxp3-deficient (scurfy) mice that reproduce the intense lymphoproliferation and allergic inflammation observed in patients with immune dysregulation polyendocrinopathy enteropathy-X-linked syndrome (41, 42). Also relevant to allergic airway disease are results from clinical studies showing the ability of Treg to down-regulate Th2 cytokine production, albeit with an apparent inability to suppress the accompanying allergic airway inflammation (15, 17). Conversely, two experimental studies using transgenic mice have suggested that Treg control allergic airway inflammation rather than AHR (13, 43), although a recent study by Lewkowich et al. (25) has indicated that Treg play a major role in the strain-specific protection of normal mice (adults) against both allergic airway inflammation and AHR. This important study however focused on the role of genetic predispositions rather than pathophysiological mechanisms related to asthma susceptibility.

Altogether, these experimental data offer limited clues to neonatal T cell and Treg function because the adult immune system is different from that of newborns, which is biased toward (Th2-driven) tolerance (44) and may (or may not) mount limited immune responses (45). The original maternal asthma model and the results presented here confirm that pups allergen sensitized at day 4 of life and allergen challenged when they are only 2 wk old can indeed develop allergic airway disease to the same extent as adults. Importantly, mAb pretreatments in early life were able to modulate this neonatal susceptibility to allergic airway disease elicited by a protocol of intentionally suboptimal asthma induction. Table I summarizes these results and the differences found in normal compared with asthma-prone pups (born of asthmatic mothers). The fact that mAb pretreatments generally led to opposing effects in normal and asthma-prone offspring indicates underlying immune differences related to maternal asthma (46).


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  		Table I. Summary of in vivo mAb treatments and their impact on early asthma susceptibilitya

 
There exist many discrepancies between in vitro and in vivo data regarding the Treg function and responsiveness (47). A major issue when studying Treg is the absence of completely specific and sensitive surface markers. The critical role for IL-2 in maintaining peripheral Treg and the fact that Treg express CD25 at very high levels has made the IL-2R {alpha}-chain a convenient (if not perfect) Treg marker, especially in untouched animals (14). A crucial experiment to address the neonatal tolerance to aeroallergens was to determine to which extent removing naturally occurring Treg from the immune system would effectively impact the pups’ susceptibility to asthma. In our hands, and as reported in adult mice (48), PC61 mAb preferentially depleted CD25high Treg with little effect on other T cells expressing low or intermediate levels of CD25. This may explain why PC61 pretreatment did not prevent the development of allergic airway disease in normal offspring. Although we cannot completely rule out the possibility that PC61 mAb also acted on activated effector T cells, the fact that extremely few CD25+Foxp3– T cells (1–2%) were found in normal and in asthma-prone pups suggests that anti-CD25 treatment mainly affected Treg in naive pups.

Flow cytometric analysis of splenic CD4+ T cell subsets showed that CD4+CD25highFoxp3+ Treg from asthma-prone offspring are phenotypically similar to those found in normal pups, at least in terms of frequencies and in terms of CTLA-4 and GITR expression. However, the baseline spleen cellularity was increased in asthma-prone offspring compared with normal pups. This was accompanied by an increase in splenic CD4+CD25–Foxp3– T cell numbers in asthma-prone offspring, a sign that T cell expansion may be less strictly controlled in pups born of asthmatic mothers. Furthermore, the unexpected amelioration of asthma susceptibility in PC61 pretreated asthma-prone pups suggested that their Treg participate in the onset of asthma. This speculation prompted us to perform complementary treatments aimed to test the hypothesis that interference with T cell and/or Treg function modulates the neonatal asthma susceptibility.

Consistent with the fact that CTLA-4 inhibits Th2 differentiation (49, 50) and participates in tolerance mechanisms (51, 52), CTLA-4 blockade with UC10-4F10 mAb exacerbated asthma syndrome in normal pups (53) without clearly ameliorating the susceptibility of asthma-prone offspring.

Conversely, GITR costimulation (DTA-1 mAb or GITR-L) reportedly abrogates Treg function, though it is unclear whether this is due to an increased T cell resistance to suppression (54), or whether it directly affects Treg (26). Our results obtained from pups treated with DTA-1 after OVA sensitization confirmed that GITR ligation in an inflammatory context (as a costimulatory signal) exacerbates allergic airway disease (37). In contrast, GITR ligation alone, in naive animals (not as a costimulatory signal) has little effect on inflammatory responses (32, 55). In this study, we found that GITR ligation in naive pups attenuates the asthma susceptibility in offspring born of asthmatic mothers, while it does not stimulate disease in normal pups. This result is particularly important because 1) it highlights intrinsic differences of T cell and Treg responses between normal and asthma-prone offspring, and 2) it suggests a distinct cascade of events when GITR cross-linking occurs before the initiation of an immune response. The idea that GITR cannot only enhance, but also restrict, T cell responses is consistent with the fact that GITR-deficient mice develop T cells that are not hypo- but hyperresponsive to CD3 cross-linking (34). Alternatively, GITR ligation in the absence of Ag presentation and other necessary signals may have induced a T cell anergy or unresponsiveness that could also explain the effects of DTA-1 pretreatment on normal and asthma-prone pups.

DTA-1 pretreatment differentially affected spleen cellularity in 7-day-old pups. It is particularly intriguing that while PC61 mAb dramatically influenced asthma susceptibility in either group of newborn mice, this pretreatment had seemingly little effect on spleen cellularity per se (aside from the removal of CD25high Treg). In contrast, DTA-1 mAb modulated both asthma susceptibility and spleen cellularity. This data suggests a cascade of events quite different from that induced by Treg removal. Moreover, it is clear that changes in CD4+CD25–Foxp3– T cell numbers could only partly account for the variations of spleen cellularity seen in DTA-1 pretreated pups. One possible explanation is that DTA-1 mAb interfered with CD4+ T cell population but also with essential interactions between various lymphocytic (CD8+ T cells, B cells) and nonlymphocytic cells (macrophages and dendritic cells) that may express GITR or its ligand (33).

Th2 cytokines, particularly IL-4 and IL-13, orchestrate the asthma syndrome (8). An important aspect of mAb pretreatment targeting T cells/Treg was to examine to what extent this would modulate cytokine production in offspring submitted to the suboptimal asthma protocol, in particular with regards to the mother’s asthma status. Multiplex analysis of BAL and serum samples showed that the cytokine production associated with the early onset of asthma is highly compartmentalized. Notably, IL-13 overexpression was found only in the lungs whereas IL-4 was overexpressed both in the lungs and at the systemic level. Also, increased IFN-{gamma} and IL-4 overproduction occurred concomitantly at the systemic level. This result is in agreement with the notion that Th1 and Th2 responses are not mutually exclusive but rather cooperate to exacerbate allergic inflammation (56, 57, 58). Given the fact that IL-4 was the only cytokine peaking both at the systemic level and in the lungs in response to suboptimal asthma induction, it is not surprising that IL-4 was also the cytokine readily modulated by mAb pretreatments.

With regard to the levels of IL-4 and IL-13 found in the lungs of asthma-prone offspring, our assumption is that mAb had an effect more potent systemically (e.g., on circulating leukocytes) than locally (e.g., on resident leukocytes and structural cells, including airway smooth muscle, respiratory epithelial cells, and fibroblasts that also produce cytokines). However, attenuation of asthma symptoms may be explained solely by abrogation of systemic IL-4 during the initiation phase of allergic airway disease (59, 60).

Treg depletion by means of PC61 mAb dramatically impacted cytokine production patterns after the pups were submitted to suboptimal asthma induction. Opposing effects were however found in normal and asthma-prone offspring. As discussed above, there remains a possibility that PC61 mAb also depleted few CD25+Foxp3– T cells present in naive pups, and/or interfered with T cell activation in the course of asthma. Nevertheless, this potential issue when using of PC61 mAb cannot explain the contrasting responses found in normal and asthma-prone offspring. Specifically, neonatal Treg depletion led to exacerbated IL-4, IL-13, and IFN-{gamma} production in normal pups submitted to the suboptimal asthma protocol. This indicates that Treg not only suppress Th1 responses (61) but are also critical to control Th2 cytokine production in normal conditions (13). Conversely, the fact that PC61 pretreatment led to decreased IL-4 and IL-13 production in offspring born of asthmatic mothers indicates that Treg, in pathological conditions, can promote local Th2 cytokine imbalance in newborn as previously observed in adult mice (62).

GITR ligation reportedly acts as a costimulus for Treg and conventional T cells activated by anti-CD3 mAb, thereby enhancing IFN-{gamma}, IL-2, IL-4, and IL-10 production (27). This costimulatory function of GITR signal may explain the exacerbation of asthma when DTA-1 mAb were injected in OVA-sensitized pups (37). However, we assume that DTA-1 mAb injected in 4-day-old naive pups had a different effect, because in these offspring IFN-{gamma}, IL-4 (serum), and IL-10 remained at (or were reduced to) low levels when the pups were submitted to suboptimal asthma induction. This likely double role for GITR signal in the modulation of T cell population and cytokine production certainly warrants further investigations (33).

Our results suggest that maternal asthma directly impacts neonatal T cell and Treg interactions. This effect may be due to a proallergic imbalance occurring during the pregnancy of asthmatic mothers (44). Indeed, it has been reported that if IL-4 does not alter the Treg function per se, it renders effector T cells less sensitive to suppression (63). Similarly, the interrelation between IL-4 and Treg function is thought to be a key event in the modulation of disease in asthmatic patients (64). Moreover, we have recently reported the existence of a cytokine imbalance, including a prominent role for IL-4 overproduction, during the pregnancy of asthmatic mother mice (18, 19). Our present finding that, despite phenotypically normal Treg, spleen cellularity is increased in naive asthma-prone offspring further supports the concept that exaggerated Th2 cytokine production during pregnancy may weaken and/or deregulate neonatal T cell/Treg interactions. This postulate warrants additional studies.

In conclusion, we demonstrate that the intrinsic asthma susceptibility seen in offspring of asthmatic mother mice can be modulated by neonatal mAb treatments. We found that early CD4+CD25highFoxp3+ Treg populations are essential to prevent the development of AHR and allergic airway inflammation in normal pups. Intriguingly, similar mAb pretreatments also revealed that offspring born of asthmatic mothers mount immune responses that may reflect dysregulated neonatal T cell/Treg interactions. Of important note, in vivo GITR ligation before allergen sensitization largely attenuated the susceptibility of asthma-prone offspring without inducing disease in normal pups. The molecular events elicited by GITR ligation as a primary signal thus bear promising therapeutic potential and merit further investigation.


   Acknowledgments
 
We thank Drs. Igor Kramnik and Mauricio Lopez-Rojas, Harvard School of Public Health, for their assistance with the xMAP technology.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 the National Institutes of Health Grant HL69760. Back

2 Current address: Momenta Pharmaceuticals, 675 West Kendall Street, Cambridge, MA 02142. Back

3 Address correspondence and reprint requests to Dr. Lester Kobzik, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. E-mail address: lkobzik{at}hsph.harvard.edu Back

4 Abbreviations used in this paper: AHR, airway hyperresponsiveness; Treg, regulatory T cell; GITR, glucocorticoid-induced TNFR family-related gene; 7-AAD, 7-aminoactinomycin D; MCh, methacholine chloride; Penh, enhanced pause; BAL, bronchoalveolar lavage. Back

Received for publication September 11, 2006. Accepted for publication November 17, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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