Lung-Infiltrating Foxp3⁺ Regulatory T Cells Are Quantitatively and Qualitatively Different during Eosinophilic and Neutrophilic Allergic Airway Inflammation but Essential To Control the Inflammation

The immune system of the lung mucosal tissues is continuously exposed to inhaled Ags, requiring regulatory mechanisms to prevent uncontrolled immune activation against otherwise innocuous Ags while mounting protective immunity against invading pathogens. Dysregulated immune responses to the harmless environmental Ags often result in asthma, a chronic inflammatory disease of the airway (1). Allergen-specific effector CD4 T cells producing Th2-type cytokines (i.e., IL-4, IL-5, and IL-13) mediate the disease processes, inducing eosinophil infiltration, IgE isotype switching, airway hyperresponsiveness, and airway remodeling (2, 3). In addition to Th2-type effector T cells, Th17-type CD4 T cells producing the signature cytokine IL-17 induce airway inflammation in which neutrophils, instead of eosinophils, are the dominant inflammatory leukocytes infiltrating the lung tissues (4, 5), and Th17-mediated neutrophilic asthma is associated with a severe persistent form (6, 7). The mechanisms underlying these distinct forms of airway inflammation remain elusive.

Foxp3⁺ regulatory T cells (Tregs) are central regulators of immunity and tolerance (8). Defects in Treg generation and/or function are coupled with uncontrolled lymphoproliferative diseases in humans and mice (8). In particular, patients with Foxp3 mutation exhibit pathologies in the mucosal tissues associated with allergic inflammation (9, 10), suggesting that Tregs are key regulators of allergic inflammation. Tregs are recruited to the inflammatory sites, where they exert regulatory functions to dampen the inflammation (11). Indeed, the proportions of Tregs are significantly elevated in bronchoalveolar lavage (BAL) fluid from asthmatic patients compared with that from healthy subjects (12). However, other investigators reported that Treg proportions are comparable between patients and healthy controls, although a lower level of Foxp3 mRNA is found in peripheral blood from asthmatics (13, 14). These conflicting results warrant further investigation with regard to the regulatory roles of Tregs during airway inflammation. Moreover, the role of lung-infiltrating Tregs during Th2-type eosinophilic and Th17-type neutrophilic airway inflammation has not formally been tested.

In this study, we examined the role of Tregs using murine models of eosinophilic and neutrophilic allergic inflammation induced via different adjuvants. We found that Treg accumulation in the inflamed lung tissues is dramatically different between the models. In eosinophilic inflammation, substantial proportions of infiltrating CD4 T cells were Foxp3⁺ Tregs, whereas the proportion was significantly lower during neutrophilic inflammation. Nonetheless, Tregs play a role in controlling both types of inflammation, because depleting Tregs during allergen challenge exacerbated the overall inflammation and inflammatory T cell responses, although the extent to which inflammatory responses were aggravated by Treg depletion was greater during eosinophilic inflammation. Phenotypic analysis of lung-infiltrating Tregs further uncovered that those Tregs from mice induced for eosinophilic inflammation display phenotypic and functional features associated with more potent suppression. Our results demonstrate that the suppressive mechanisms expressed by infiltrating Tregs may be shaped by environmental cues available to those Tregs infiltrating the inflamed tissues.

C57BL/6 (B6) and C57BL/6 Foxp3.DTR mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 Foxp3.GFP-knockin mice were reported previously (15). All mice were maintained in a specific pathogen–free facility located in the Lerner Research Institute. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee.

For eosinophilic airway inflammation, mice were injected i.p. with 5 μg of cockroach Ag (CA; Greer Laboratory, Lenoir, NC) mixed in 100 μl of alum adjuvant (aluminum hydroxide; Sigma, St. Louis, MO). Another injection was given 7 d later. Starting on day 14, the mice were challenged daily with an intranasal CA injection (5 μg in 50 μl) for 4 d. Mice were sacrificed 24 h after the last Ag challenge. For neutrophilic airway inflammation, mice were immunized s.c. with 5 μg of CA emulsified in 100 μl of CFA containing 5 mg/ml H37Ra (BD Difco, Franklin Lakes, NJ). Starting on day 14, the mice were challenged with CA and sacrificed similarly to the eosinophilic inflammation model. In some experiments, Tregs were depleted by i.p. injection of 1 μg of diphtheria toxin (DTX; Sigma). Lung tissues were prepared from paraffin-embedded blocks and stained with H&E. Inflammation was scored as previously reported (16): 0, no or occasional cells; 1, one or two concentric rows of perivascular and peribronchial cell accumulation; 2, three or more concentric rows of perivascular and peribronchial cell accumulation; and 3, continuous perivascular and peribronchial cell accumulation.

At sacrifice, BAL fluid cells were collected. Lung tissue was minced with scissors, and a single-cell suspension was obtained. Mononuclear cells were isolated from the 30/70% Percoll interface. RBCs were lysed with ACK lysis buffer. Cells were stained with anti-CD4 (RM4-5), anti-Ly6G (1A8), anti–Siglec-F (1RNM44N), anti-ICOS (C398.4A), anti-CD39 (24DMS1), anti–glucocorticoid-induced TNFR (GITR) (DTA-1), anti-ICOS ligand (ICOSL; HK5.3), anti-CD25 (PC61), anti-CD31 (MEC13.3), anti-Vcam1 (429), anti-Nrp1 (3DS304M), anti-CD11c (N418), anti-CD11b (M1/70), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-BrdU (3D4), and anti-CD49d (R1-2). For intracellular staining, cells were stained with anti–Ki-67 (B56), anti–IL-4 (11B11), anti–IL-10 (JES5-16E3), anti–IL-13 (eBio13A), anti–IL-17 (TC11-18H10), and anti–IFN-γ (XMG1.2). All of the Abs used in this study were purchased from eBioscience (San Diego, CA), BD Pharmingen (San Jose, CA), and BioLegend (San Diego, CA). Cells were acquired using an LSR II (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR). For intracellular staining, cells were harvested separately and stimulated ex vivo with PMA (10 ng/ml) and ionomycin (1 μM) for 4 h in the presence of 2 μM monensin (Calbiochem, San Diego, CA) during the last 2 h of stimulation. Cells were immediately fixed with 4% paraformaldehyde, permeabilized, and stained with fluorescence-conjugated Abs. In some experiments, mice were injected with 1 mg of BrdU (Sigma) 24 h prior to sacrifice. In vivo BrdU incorporation was measured using a BrdU kit (BD Pharmingen), according to the manufacturer’s instructions.

Foxp3⁺ Tregs were FACS sorted from inflamed lung tissues using a FACSAria sorter (BD Biosciences). Naive responder CD4 T cells were sorted from B6 mice, labeled with CFSE, and cultured with anti-CD3 mAb plus irradiated T cell–depleted splenocytes for 3 d. Different numbers of Tregs were added to the culture. CFSE dilution was determined by FACS analysis. Relative proliferation was calculated based on CFSE dilution without Tregs.

Draining mediastinal lymph node (medLN) cells isolated from animals induced for eosinophilic and neutrophilic airway inflammation were stimulated in vitro with 10 μg/ml CA or OVA protein for 3 d. Culture supernatant was collected, and IL-4/IL-17 secretion was determined by ELISA. In some experiments, BAL fluids were collected from mice induced for eosinophilic and neutrophilic inflammation and treated with PBS or DTX. IL-4 and IL-17 secretion was determined by ELISA.

An assay for Mouse Inflammation Array C1 (code AAM-INF-1-4; RayBiotech, Norcross, GA) was carried out according to the manufacturer’s instructions. Briefly, Ab-coated array membranes were incubated for 30 min with 1 ml of blocking buffer. After 30 min, blocking buffer was decanted and replaced with 1 ml of the samples at 4°C overnight. The samples were decanted from each container and washed three times with wash buffer at room temperature with shaking for 10 min. After extensive washing to remove unbound materials, membranes were then incubated with diluted biotin-conjugated Abs at 4°C overnight. Membranes were washed and then incubated with HRP-conjugated streptavidin at room temperature for 2 h. Membranes were then washed thoroughly and exposed to detection buffer in the dark before being exposed to x-ray film, and images were developed using a film scanner. Intensities of signals were quantified using ImageJ software. Positive controls were used to normalize results from different membranes being compared. Fold changes in protein expression were calculated.

RNA was isolated from inflamed lung tissues or FACS-sorted cells using a GeneJET RNA isolation kit (Thermo Fisher, Waltham, MA), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega, Madison, WI). Real-time PCR analysis was performed using an ABI 7500 Real-Time PCR system, SYBR master mix (both from Applied Biosystems, Waltham, MA), and gene-specific primers.

Hierarchical clustering and heat map generation were performed using nSolver Software. Statistical significance was determined by the t test (two-tailed) using Prism 5 software (GraphPad, La Jolla, CA). A p value <0.05 was considered statistically significant.

Using CA as a model allergen, we employed alum- and CFA-based sensitization protocols known to induce eosinophilic and neutrophilic airway inflammation, respectively (17–20). Mice were sensitized with CA in alum and intranasally challenged with CA (Supplemental Fig. 1). When measured 24 h after the last challenge (day 5), they developed eosinophilic inflammation in the airway that was characterized by predominant SiglecF⁺ (Ly6G⁻) eosinophil infiltration in the BAL fluid (Fig. 1A). Approximately 90% of infiltrating cells were eosinophils, <1% were neutrophils, and CD4 T cells constituted <5% (Fig. 1B). In contrast, mice sensitized with CA in CFA, followed by intranasal allergen challenge, developed airway inflammation with predominantly neutrophils (Ly6G⁺SiglecF⁻) in the BAL fluid (Fig. 1A, Supplemental Fig. 1). Approximately 50% of the infiltrating cells were neutrophils, whereas <10% were eosinophils (Fig. 1B). Eosinophil-dominant BAL fluid cell profiles remained consistent before and after the peak responses (Fig. 1C, Supplemental Fig. 2). In CFA-induced inflammation, neutrophils were dominant at days 5 and 7, although their proportions and numbers gradually decreased after day 7 (Fig. 1C, Supplemental Fig. 2). CD4 T cells expressing Th2 and Th17 signature cytokines are the mediators of eosinophilic and neutrophilic inflammation, respectively (5, 7, 21). Indeed, alum-induced inflammation was strongly associated with Th2-type effector CD4 T cells in the draining lymph node (LN) and lung tissues. T cells expressing Th2-type cytokines (i.e., IL-4, IL-13, and IL-10) were more abundant compared with T cells expressing IFN-γ or IL-17 (Fig. 1D, 1E). Likewise, IFN-γ– and IL-17–producing effector CD4 T cells were significantly elevated in mice induced for neutrophilic inflammation, whereas T cells expressing Th2-type cytokines were substantially lower compared with those from alum-induced inflammation (Fig. 1D, 1E). Th2- and Th17-type responses were Ag specific, because draining LN CD4 T cells from alum- and CFA-sensitized mice primarily produced IL-4 and IL-17, respectively, upon restimulation with CA Ags but not with irrelevant Ags (Fig. 1F). Taken together, these results demonstrate two distinct inflammatory responses in the airway characterized by Th2/eosinophils or by Th1/Th17/neutrophils.

Foxp3⁺ Tregs accumulate in inflamed tissues, where they are thought to exert regulatory functions (22, 23). There is increasing evidence that Treg functions can be compromised during allergic inflammation and that such defective functions contribute to disease aggravation (13, 24). To investigate tissue-infiltrating Tregs in eosinophilic and neutrophilic inflammation, Foxp3^GFP reporter animals were induced for inflammation, and Treg accumulation in the inflamed lung tissues at the peak of the responses (24 h after the last challenge, day 5) was compared. We found that ∼30% of CD4 T cells infiltrating the BAL fluid and lung tissues of mice induced for eosinophilic inflammation expressed GFP, whereas Treg infiltration to the inflamed tissues during neutrophilic inflammation were only ∼10% among total CD4 T cells (Fig. 2A). The differential Treg accumulation was only found in the inflamed tissues, because Treg levels in the draining medLN and other lymphoid tissues were comparable between the groups (Fig. 2A, data not shown).

To ensure that differential Treg accumulation in the inflamed tissues in the aforementioned experiments is not due to different kinetics of Treg trafficking, we compared Treg infiltration during and after Ag challenge. Four time points were chosen, as described in Supplemental Fig. 1: 1 d after the second CA challenge (day 3) and 1 d (day 5), 3 d (day 7), and 6 d (day 10) after the last CA challenge. In the case of alum-induced inflammation, Treg accumulation in the target tissues (BAL fluid and lung) was already elevated at day 3, peaked around day 5, and gradually diminished thereafter (Fig. 2B). In contrast, Tregs remained at low levels throughout the experiments in CFA-induced inflammation (Fig. 2B). Again, Treg levels in the draining LN were comparable at all times points examined in both groups (Fig. 2B). Interestingly, the ratio of Tregs/effector CD4 T cells was drastically different between the two groups. As shown in Fig. 2C, effector T cells significantly outnumbered Tregs in the lung of CFA-sensitized mice. In contrast, the ratio of effector T cells/Tregs remained relatively low in alum-sensitized mice (Fig. 2C). The ratio remained constant in the draining LN, regardless of the mode of sensitization (Fig. 2C).

Thus far, our results demonstrate that Treg accumulation in the inflamed lung tissues is differentially controlled by the nature of airway inflammation (i.e., eosinophilic versus neutrophilic inflammation). The role of Tregs during allergen-induced airway inflammation has been examined previously. Using bacterial artificial chromosome–transgenic Foxp3.DTR (DEREG) mice, Baru et al. (25) reported that the lack of Tregs during OVA Ag challenge does not aggravate allergic airway inflammation. We tested the roles of Tregs during CA-induced eosinophilic inflammation using Foxp3.DTR-knockin mice (26). Following CA sensitization in alum, DTX was injected 1 d prior to and on the first day of CA challenge (Fig. 3A). Tregs were efficiently ablated when analyzed 24 h after the last CA challenge (Fig. 3B). Eosinophils were still the dominant population in the BAL fluid after Treg depletion, although the absolute numbers of lymphocytes/macrophages and neutrophils were significantly elevated (Fig. 3C). However, unlike the previous report (25), we found that Treg depletion during allergen challenge dramatically exacerbated the lung inflammation, as determined by H&E (Fig. 3D) and periodic acid–Schiff staining (data not shown). In support of periodic acid–Schiff staining, Muc5a and Muc5b mRNA expression was significantly elevated in the lung of Treg-depleted animals (Fig. 3E). The levels of lung-infiltrating CD4 T cells producing Th2-type cytokines were similarly elevated upon Treg depletion (Fig. 3F). We estimated an ∼10-fold increase in effector T cells expressing Th2-type cytokines in the lung (Fig. 3F). IL-17–producing CD4 T cells were also increased ∼3-fold following depletion (Fig. 3F), which may account for increased neutrophil infiltration (Fig. 3C). The magnitude of increase in cytokine-producing cells was even greater in the draining medLN (Fig. 3G). Consistent with intracellular cytokine expression, we detected increased IL-4 secretion in the BAL fluid after Treg depletion (Fig. 3H). Altered inflammatory responses following DTX-mediated Treg depletion were not observed in B6 non-DTR mice (Supplemental Fig. 3), suggesting that the enhanced inflammatory responses are not off-target effects caused by the potential adjuvant effect of injected DTX. Taken together, these results strongly suggest that Foxp3⁺ Tregs play an essential role in controlling eosinophilic airway inflammation during allergen challenge.

Treg accumulation in the lung tissues during CFA-induced neutrophilic airway inflammation was substantially lower than that during eosinophilic inflammation (Fig. 2A). Whether these Tregs still play a role in controlling inflammatory responses in this condition was tested next. Analogous to the alum-induced model (Fig. 3), DTX was injected into Foxp3.DTR mice sensitized with CA in CFA adjuvant 1 d prior to and on the first day of CA challenge (Fig. 4A). Complete depletion of Tregs was confirmed at sacrifice (Fig. 4B). Without Tregs, neutrophils remained the dominant population in the BAL fluid; however, the overall cellularity of inflammatory cells in the BAL fluid was significantly elevated (Fig. 4C). Lung inflammation was aggravated following Treg depletion (Fig. 4D), suggesting that Tregs, albeit at a lower proportion, still play an important regulatory role. Likewise, Muc5a and Muc5b mRNA expression was elevated (Fig. 4E). Notably, Treg depletion during neutrophilic inflammation increased the numbers of T cells expressing Th2-type cytokines (IL-4 and IL-13), although the magnitude of the increase was considerably lower than that seen during alum-induced inflammation (Fig. 4F, 4G). The accumulation of IL-17–expressing CD4 T cells remained unchanged when measured by intracellular cytokine staining; however, IL-17 secretion in the BAL fluid was significantly increased following Treg depletion (Fig. 4F–H). IFN-γ⁺ CD4 T cell accumulation was significantly increased by Treg depletion. Therefore, these results suggest that Tregs still play a key regulatory role during CFA-induced neutrophilic airway inflammation, although their lung accumulation is less evident. It is important to point out that the magnitude of enhanced inflammation following Treg depletion is greater during eosinophilic inflammation (Fig. 3F, 3G) than during neutrophilic inflammation (Fig. 4F, 4G), suggesting that the extent to which Tregs control inflammatory responses may be different depending on the inflammatory environments.

Allergen-specific CD4 T cells upregulate the α4 integrin (CD49d) that facilitates T cell migration to the sites of inflammation (27, 28). CD49d upregulated on activated T cells forms the α4β1 integrin complexes (VLA-4), which then bind to fibronectin and VCAM-1 for lung infiltration during airway inflammation (29). Proper control of Treg migration is an essential feature for successful Treg functions in vivo (30). Thus, we examined whether CD49d is differentially expressed by lung-infiltrating Tregs during alum- and CFA-induced inflammation. CD49d⁺ Foxp3⁺ Tregs within the inflamed tissues were significantly greater in alum-induced inflammation than in CFA-induced inflammation, whereas LN-resident Tregs expressed comparable levels of CD49d (Fig. 5A). Interestingly, CD49d expression on effector CD4 T cells in the lung was comparable (Fig. 5A). The expression of VCAM-1, the ligand for CD49d, on lung endothelial cells was comparable between the groups (Fig. 5B). In addition to tissue-trafficking adhesion molecules, inflammatory chemokine expression may play a role in differentially attracting Tregs during alum- and CFA-induced inflammation. Measuring chemokine expression in the lungs by protein array analysis revealed distinct chemokine-expression patterns in alum- and CFA-induced lung inflammation (Fig. 5C). In alum-induced inflammation, higher levels of CXCL12 and CCL5 expression were observed, whereas CXCL9, CCL25, CXCL13, CCL24, and CXCL1 expression was significantly increased in the lungs with CFA-induced inflammation (Fig. 5C). Tregs migrate to the airways via CCR4 and attenuate allergic inflammation (31), and expression of the CCR4 ligands, CCL17 and CCL22, is essential in recruiting Tregs to the inflamed lung during allergen challenge (32). However, we found that CCR4 expression in infiltrating Tregs and the CCR4 ligand expression in the lung tissues in both models remained comparable (data not shown), suggesting that the CCL17/CCL22-CCR4 axis plays little role during elevated Treg recruitment in alum-induced inflammation.

Although Treg depletion in the alum- and CFA-induced models resulted in aggravation of lung inflammation (Figs. 3, 4), we noted that the extent to which inflammatory T cell responses are enhanced by Treg depletion was especially greater in the alum-induced model, raising the possibility that the mechanism by which Tregs control the inflammation may be different. The expression of surface molecules associated with suppressive functions on the lung-infiltrating Tregs was measured. ICOS is a marker for highly suppressive Ag-specific Tregs, in part by controlling Treg expansion during helminth infections (33, 34). Indeed, lung-infiltrating Tregs expressed high levels of surface ICOS (Fig. 6A). The proportion of ICOS⁺ Tregs was comparable between the groups; however, the mean fluorescence intensity (MFI) of ICOS expression was reproducibly higher in Tregs from CFA-sensitized mice (Fig. 6A). ICOS⁺ Tregs also expressed Nrp1, a thymus-derived Treg–specific marker implicated in Treg stability (35, 36). However, unlike ICOS, the MFI of Nrp1 expression was greater in Tregs from alum-sensitized mice (Fig. 6A). Unexpectedly, we noticed that a substantial proportion of Foxp3⁻ lung-infiltrating effector CD4 T cells also expressed Nrp1, which also expressed ICOS (Fig. 6A). It appears that these Nrp1⁺ Foxp3⁻ CD4 T cells are highly activated effector T cells, but not a subset of Tregs, because they were highly proliferating BrdU⁺ cells and dramatically increased following Treg depletion (Q.T. Nguyen and B. Min, unpublished observations). The proportions of Tregs expressing GITR and CTLA4 were comparable between the groups (Fig. 6B, data not shown). CD39, an ectoenzyme that hydrolyzes ATP, is highly expressed in Tregs at the site of inflammation (37, 38). We found that Tregs expressing CD39 were more abundant in the lung tissues than in the lymphoid tissues (data not shown) and that the level was higher in CFA-sensitized mice (Fig. 6B). Therefore, Tregs infiltrating the lung tissues may mediate regulatory functions via different mechanisms, depending on the types of inflammation.

Costimulatory signals are critical to control Treg functions (39). We next compared APC expression of costimulatory ligands implicated in controlling Treg functions. We found that CD80 and CD86 expression was also higher during alum-induced inflammation models (Fig. 6C). ICOSL expression remained comparable (Fig. 6C).

Tregs are a highly proliferative population in vivo, and ∼20% of LN Tregs are shown in active cell cycle (40, 41). Tregs in the lung tissues expressed high levels (∼80%) of Ki-67, which is in stark contrast to the proportion of cycling Tregs in the medLN (30– 40% Ki-67⁺) (Fig. 7A). The proportions of cells in the active cell cycle were similar, regardless of the type of inflammation (Fig. 7A). However, when the rate of cell proliferation was determined by in vivo BrdU incorporation, which only detects the S phase of the cell cycle, over a 24-h period, BrdU incorporation was significantly higher in Tregs from CFA-sensitized mice, suggesting that more Tregs divide in CFA-induced inflammation (Fig. 7A). However, these Tregs expressed higher active caspase 8 (Fig. 7A). Therefore, Tregs differentially infiltrating the inflamed lung tissues during eosinophilic and neutrophilic airway inflammation display distinct homeostatic behaviors. Of note, the Foxp3 expression level was comparable (Fig. 7B).

It was reported previously that the Treg accumulation pattern in the inflamed skin is linked to CD25 expression (42). Consistent with this study, we found that Tregs infiltrating the lung tissues during alum-induced inflammation expressed significantly higher surface CD25 (Fig. 7C). Furthermore, those Tregs expressed more potent suppressive activity compared with Tregs from the CFA-sensitized groups (Fig. 7D). Treg-mediated control of tissue inflammation can be changed dynamically by inflammatory signals (43). In particular, Gata3, a Th2 lineage transcription factor, plays a crucial role in regulating Treg accumulation in the inflamed sites and maintaining Foxp3 expression (44). Indeed, Treg expression of Gata3 was dramatically higher in Tregs during alum-induced inflammation (Fig. 7E, 7F), whereas the expression of other lineage-specific transcription factors, such as T-bet or RORγt, remained comparable (Fig. 7F). Furthermore, these Tregs also expressed higher IL-10 (Fig. 7F), further supporting more potent suppressive capacity, as well as increased accumulation in the inflamed lung tissues. Taken together, Tregs recruited into the inflammatory sites during eosinophilic airway inflammation appear to express key molecules that promote the suppressive functions, possibly in response to inflammatory signals available to them.

Accumulation of Foxp3⁺ Tregs in inflamed tissues is a critical regulatory mechanism by which inflammatory responses are resolved (11). In this article, we report that Treg accumulation in the lung tissues during allergic airway inflammation is differentially controlled by the types of inflammation (i.e., eosinophilic versus neutrophilic). In the alum adjuvant–induced inflammation model in which eosinophils and Th2-type effector CD4 T cells are the dominant inflammatory cell types, substantial (20–30%) proportions of lung-infiltrating CD4 T cells are Tregs. In contrast, the proportion of lung-infiltrating Tregs is merely ∼10% during CFA adjuvant–induced airway inflammation in which neutrophils and Th1/Th17-type effector CD4 T cells are predominantly found. The proportions of Tregs in the draining LN remain comparable between the two model systems, suggesting that different levels of Treg accumulation within the inflamed tissues are likely due to differential recruitment, expansion, survival, or any combination thereof, resulting in different regulatory mechanisms to control inflammation.

Faustino et al. (45) reported that Tregs expressing effector/memory phenotypes accumulate in the lung of allergic animals and that these Tregs efficiently suppress pulmonary T cell proliferation but not Th2 cytokine production. Baru et al. (25) reported that the absence of Tregs during OVA allergen challenge does not exacerbate allergic airway inflammation. Our results clearly demonstrate that accumulation of effector CD4 T cells expressing inflammatory cytokines in the lung tissues is dramatically elevated upon Treg depletion during allergen challenge in both models of lung inflammation, suggesting that Tregs play a regulatory role in controlling Th2- and Th1/17-type airway inflammation during allergen challenge. The difference behind the discrepancy is unclear. Unlike OVA, which is typically used, our study used CA, which is capable of activating protease-activated receptors (46). Protease-activated receptors enhance production of inflammatory cytokines and potentiate Th2-type responses (47). The model allergen may affect the role of Tregs during allergic inflammation.

Fowell and colleagues (42) reported that the magnitude of Treg accumulation in the inflamed skin varies depending on the adjuvants used to immunize animals. They similarly found that Treg accumulation after alum-induced skin inflammation is greater than that induced by CFA immunization (42). The Treg accumulation pattern was found to be linked to CD25 expression in Tregs (42). Consistently, we found that Tregs infiltrating the lung tissues from alum-induced inflammation expressed higher CD25. One potential modulator that controls infiltrating Treg functions is APCs. It was noted that APC expression of costimulatory molecules, such as CD80/CD86, is directly linked to Treg expansion/accumulation (42). We also found that the expression of CD80 and CD86 was significantly upregulated, especially in CD11c⁻ CD11b⁺ macrophages in the lung during eosinophilic airway inflammation, possibly supporting the importance of the CD28/B7 pathway for the homeostasis of Tregs to control inflammatory responses and to maintain CD25 expression on Tregs (48, 49). During CFA- and alum adjuvant–induced skin inflammation, the expression of inflammatory signature genes remained unchanged in Tregs (42). However, we found that Gata3 expression is considerably elevated in Tregs following alum-induced airway inflammation. Supporting these findings is the fact that Gata3 expression in Foxp3⁺ Tregs critically controls Treg accumulation at inflamed barrier sites and that Gata3 expression by Tregs is positively regulated by IL-2 (44).

The findings that the magnitude of aggravation of inflammatory responses following Treg depletion is different between eosinophilic and neutrophilic inflammation raises an interesting possibility that the regulatory functions of Tregs may be controlled at sites of inflammation. As previously proposed (43), inflammatory factors produced by effector cells may be capable of altering transcription factor expression (e.g., Stat3, IRF4, and Gata3) in Tregs, from which Tregs may acquire distinct regulatory mechanisms to control inflammatory responses. Particularly interesting is that Treg depletion during eosinophilic inflammation enhances effector CD4 T cells producing Th2-type, as well as Th17-type, cytokines. In contrast, IL-17 production remains unchanged following Treg depletion during neutrophilic inflammation. Instead, significant increases in T cells producing Th2-type cytokines and IFN-γ are observed in this condition, although the levels are still significantly lower than those found in eosinophilic inflammation. Understanding the precise nature of cross-talk between infiltrating Tregs and inflammatory factors and the specific regulatory functions that Tregs carry out under such conditions are the subjects of future investigation.

In conclusion, the current results demonstrate that the magnitude of Treg accumulation in inflamed lung tissues may still be an important factor that mediates Treg functions to control inflammation. However, Treg-extrinsic factors from the inflammatory milieu and APCs may be equally important in shaping Treg functions.

We thank Jennifer Powers for cell sorting.

The authors have no financial conflicts of interest.