Research ArticleALLERGY

PPAR-γ promotes type 2 immune responses in allergy and nematode infection

See allHide authors and affiliations

Science Immunology  10 Mar 2017:
Vol. 2, Issue 9, eaal5196
DOI: 10.1126/sciimmunol.aal5196

Type 2 immunity on PPAR

Agonists to the anti-inflammatory molecule peroxisome proliferator activated receptor–γ (PPAR-γ) decrease airway inflammation in animal models but have had limited success in clinical trials. Now, Chen et al. examine the contribution of PPAR-γ to T helper type 2 (TH2) immunity, which is critical for both allergy and parasite immune response. Mice that lacked PPAR-γ did not develop allergic pathology and failed to protect against nematode infection. These mice lacked interleukin-5 (IL-5)– and IL-13–secreting cells and had reduced frequencies of TH2 cells in visceral adipose tissue. PPAR-γ agonists up-regulated the IL-33 receptor ST2. Hence, PPAR-γ may contribute to IL-5 and IL-13 production by TH2 cells, which highlights the need for caution in using PPAR-γ agonists for TH2-mediated diseases.

Abstract

A hallmark of immunity to worm infections and many allergies is a strong type 2 immune response. This is characterized by the production of cytokines interleukin-5 (IL-5) and IL-13 by adaptive T helper 2 (TH2) cells and/or type 2 innate lymphoid cells. Peroxisome proliferator activated receptor–γ (PPAR-γ) is typically regarded as an anti-inflammatory factor. We report that TH2 cells express high levels of PPAR-γ in response to the allergen house dust mite and after infection with the parasite Heligmosomoides polygyrus. Mice lacking PPAR-γ in T cells failed to effectively differentiate into IL-5– and IL-13–secreting cells and, hence, did not develop TH2 cell–associated pathologies, including goblet cell metaplasia and eosinophilia, in response to allergen challenge. Furthermore, these mice could not mount protective immune responses to nematode infection. In addition, mice lacking PPAR-γ in T cells had greatly reduced frequencies of TH2 cells in visceral adipose tissue. Mechanistically, PPAR-γ appeared to promote the expression of the IL-33 receptor on the surface of TH2 cells. These results pinpoint PPAR-γ as a factor that drives type 2 responses in allergy, worm infection, and visceral adipose tissue.

INTRODUCTION

Naïve CD4 T cell activation initiates the differentiation of these cells into mature, functionally specialized T helper (TH) cell subsets. To date, a number of TH cell subsets have been delineated, including TH1, TH2, TH17, and suppressive regulatory T cells (Tregs) (1, 2). Analogous to adaptive TH cells, innate lymphoid cells (ILCs) have been shown to develop from a common bone marrow precursor into ILC1, ILC2, and ILC3, which share features of TH1, TH2, and TH17 cells, respectively (3). Type 2 immune responses, which are mediated by TH2 cells and ILC2, are essential for immunity to large extracellular pathogens such as worms. TH2 cells and ILC2 are characterized by the production of cytokines interleukin-4 (IL-4), IL-5, and IL-13, which promote macrophage functions, epithelial cell activation, and B cell immunoglobulin E (IgE) secretion (1). Although type 2 responses are beneficial in some contexts, these responses are heavily implicated in allergies such as asthma, whose incidence has risen markedly in the past few decades (4).

The differentiation of naïve CD4 T cells into functional TH2 cells depends on many environmental cues and cell-intrinsic changes. Typically, IL-4 from the extracellular environment promotes activation of signal transducer and activator of transcription 6 (STAT-6), which rapidly induces the master transcriptional regulator of TH2 cells, GATA-3 (1, 5). Together, GATA-3 and STAT-6 promote changes to the chromatin architecture of differentiating cells, support proliferation, and promote the transcription of effector cytokine genes, including IL-4, IL-5, and IL-13 (1, 6, 7).

An important function of GATA-3 is to promote the expression of the IL-33 receptor (IL-33R) on the surface of differentiating TH2 cells (8). The IL-33R, which is a hallmark of TH2 cells, is composed of a common IL-1RAP chain and the subunit ST2 and signals through MyD88 to promote nuclear factor κB activation and stimulate the production of effector cytokines (1, 9). Expression of the IL-33R on the surface of TH2 cells is critical for the function of these cells (10, 11), and single-nucleotide polymorphisms (SNPs) in the IL33 or IL1RL1 (ST2) genes are strongly associated with an increased risk of asthma (12) and heart disease (13). A recent study suggested that the acquisition of full effector functions by TH2 cells and ILC2 depended on their migration to organs (such as the lung) and exposure to cytokines (such as IL-33, IL-25, and thymic stromal lymphopoietin) (14). Thus, the priming lymph node is not sufficient for fulminant TH2 cell responses, and the elucidation of tissue-specific factors that regulate responsiveness to the cytokine IL-33, for example, could lead to substantial improvements in the treatment of many inflammatory disorders.

The nuclear receptor peroxisome proliferator activated receptor–γ (PPAR-γ) is a master regulator of adipocyte differentiation and a potent modulator of lipid metabolism (15). Studies over the past few decades have demonstrated that PPAR-γ suppresses the secretion of proinflammatory cytokines by epithelial cells (16, 17), promotes type 2 macrophage development (18), and enhances Treg accumulation at inflammatory sites such as the visceral adipose tissue (VAT) (19). This strong anti-inflammatory function has suggested that agonists of PPAR-γ, known as thiazoldinediones (TZDs), may be useful for the treatment of various disorders (20). To this end, TZDs have repeatedly shown strong therapeutic efficacy in preclinical models of asthma (21, 22), and this has led to early-phase clinical trials in this setting (23).

In this study, we identified PPAR-γ as a gene selectively up-regulated in TH2 cells after exposure to house dust mite (HDM) extracts or the nematode Heligmosomoides polygyrus. In contrast to the known anti-inflammatory function of PPAR-γ, TH2 cells were dependent on PPAR-γ for their differentiation into IL-5– and IL-13–producing effector cells. PPAR-γ expression in CD4 T cells promoted pathogenic TH2 cell responses to HDM and protective type 2 immunity to H. polygyrus. Mice lacking PPAR-γ in T cells were unable to effectively up-regulate the IL-33R subunit ST2. These results identify PPAR-γ as a crucial amplifier of type 2 immune responses.

RESULTS

TH2 cells preferentially express PPAR-γ

We recently performed whole-genome expression profiling on TH cells isolated from the lungs of mice sensitized and challenged with HDM extracts (24). A strong up-regulation in the expression of Pparg and several known PPAR-γ target genes, including Retnla, Arg1, Chi3l3, and Chi3l4, was observed (25), implying that this nuclear receptor was driving the response (Fig. 1A). Because HDM induces both TH2 and TH17 cell populations in the mouse lung, we sought to clarify which subset was expressing PPAR-γ. The surrogate markers ST2 and CCR6 were used to identify TH2 and TH17 cells, respectively, from the CD4+ T cell population (10, 26). Pparg mRNA was preferentially expressed by the ST2+ cell fraction (P < 0.001) expressing high levels of IL-5 (Fig. 1, B to E). TH2 cells also play a critical protective role in immunity to helminth infection (27). To examine whether a similar pattern of PPAR-γ expression was observed in mice infected with the nematode H. polygyrus, we purified TH2 (ST2+), TH17 (CCR6+), and TH1 cells [which express the chemokine receptor CXCR3 (28)] from the mesenteric lymph nodes (mesLN) of infected mice. Quantitative polymerase chain reaction (PCR) confirmed that ST2+ cells expressed the most Il5 (P < 0.001), CCR6+ cells expressed the most Il17a (P < 0.001), and CXCR3+ cells expressed the highest levels of Ifng mRNA (P < 0.05) (Fig. 1, H to J). Levels of Pparg mRNA were again more abundantly expressed in TH2 cells after worm infection (P < 0.001) (Fig. 1G). Thus, PPAR-γ appears to be preferentially expressed by TH2 cells in the two models where type 2 immunity is important. We further characterized the expression of PPAR-γ after classical TH cell differentiation cultures. Similarly, Pparg mRNA was most highly expressed in cells cultured under TH2 cell differentiation conditions (Fig. 1K), and this expression appeared dependent on the master transcriptional regulator GATA-3 because loss of one allele of Gata3 reduced Pparg mRNA levels significantly (P = 0.0286) (Fig. 1L). These results demonstrate that PPAR-γ is a TH2 cell–specific factor in multiple contexts.

Fig. 1 PPAR-γ is expressed specifically in TH2 cells.

(A) Genome-wide expression analysis of CD4+ T cells (CD4+CD45+F4/80) purified from the lungs of naïve PBS-treated or HDM-sensitized and HDM-challenged mice on day 15. Clustering analysis of PPAR-γ (indicated by a red arrow) and a number of its targets is depicted. Values are log2-transformed. (B to E) Il5, Il17a, and Pparg mRNA levels in purified TH2 (ST2+), TH17 (CCR6+ST2), and TH0 cells from the lungs of HDM-sensitized and HDM-challenged mice. (F to J) Il5, Il17a, Ifng, and Pparg mRNA levels in purified TH2 (ST2+), TH17 (CCR6+ST2CXCR3), TH1 (CXCR3+ST2), and TH0 cells from mesLN of H. polygyrus–infected mice 15 days after infection. Data are representative of two independent experiments with n = 3 to 4 mice in each experiment (means and SEM are depicted). N.D., not done. In (C) to (J), all subsets are compared with the TH0 subset. (K) Naïve CD4 T cells were differentiated into TH1, TH2, TH17, and Treg cells, and the expression of Pparg mRNA was analyzed. (L) Pparg mRNA expression in WT Gata3+/+ and heterozygous Gata3+/− TH2 cells in vitro (n = 4 separate mice for each genotype from one of two representative experiments). (C) to (J) were analyzed using one-way ANOVA and Bonferonni’s test for multiple comparisons to the TH0 subset. Mann-Whitney U test was used in (L). *P < 0.05, **P < 0.01, and ***P < 0.001.

PPAR-γ expression in T cells drives pathogenic type 2 immune responses to HDM

To determine the effect of PPAR-γ on TH2 cell function, we crossed mice in which LoxP sites were engineered into the Pparg gene with mice expressing the Lck-Cre transgene. Mice in which T cells were targeted for deletion of the Pparg gene (Lck-Cre+Ppargfl/fl) are termed PpargLckΔ here, and wild-type (WT) littermate control Lck-CrePpargfl/fl mice are referred to as Pparg+. We first investigated the presence of alveolar macrophages in the airways because PPAR-γ is essential for their development (25). Normal levels of alveolar macrophages were observed in bronchoalveolar lavage (BAL) samples of PpargLckΔ mice administered phosphate-buffered saline (PBS) (Fig. 2, A and B), suggesting that Pparg was not deleted in this myeloid lineage. To test whether Pparg was deleted in CD4 T cells, we activated naïve CD4 T cells in the presence of TH2 cell differentiation factors and analyzed the expression of Pparg mRNA after 4 days. Levels of Pparg transcripts were reduced by about 90% in CD4 T cells from PpargLckΔ mice, confirming that the Pparg gene was excised in the CD4 T cell lineage (Fig. 2C). To determine the impact of PPAR-γ deletion on TH2 cell responses in vivo, we sensitized and challenged mice with HDM extracts and analyzed the composition of airway-infiltrating cells and mucus secretion, two hallmarks of the asthmatic response. After administration of HDM, PpargLckΔ mice had reduced frequencies (P = 0.0013) and numbers (P = 0.0004) of airway eosinophils compared with control Pparg+ mice (Fig. 2, D to F). In addition, PpargLckΔ mice had reduced infiltration of T cells into the airways but normal levels of alveolar macrophages and neutrophils (P = 0.0149) (Fig. 2G). Furthermore, periodic acid–Schiff staining of lung sections revealed a complete absence of mucus-secreting goblet cells lining the airways of PpargLckΔ mice (Fig. 2H). This strongly suggested that PPAR-γ expression in CD4 T cells was driving the pathogenic hallmarks of the asthmatic response and that PPAR-γ was important for the differentiation of TH2 cells.

Fig. 2 PPAR-γ promotes pathogenic responses to HDM.

(A) Representative plots of CD11c versus Siglec-F expression in BAL of naïve PBS-treated WT littermate control Lck-CrePpargfl/fl (Pparg+, n = 3) and in Lck-Cre+Ppargfl/fl (PpargLckΔ, n = 4) mice. (B) Frequency of alveolar macrophages (Al. mφ) shown in the top right quadrant of (A). (C) Relative expression of Pparg mRNA from naïve CD4 T cells activated under TH2 cell differentiation conditions for 4 days (control Pparg+, n = 6; PpargLckΔ, n = 8). (D) Representative plots of CD11c versus Siglec-F expression in BAL samples from control Pparg+ and PpargLckΔ mice sensitized and challenged with HDM on day 15 (frequency of SiglecF+CD11c eosinophils is indicated). (E and F) Graphs of BAL eosinophil (Siglec-F+CD11c) frequency and number in PpargLckΔ (n = 12) compared with WT control mice (Pparg+, n = 17) sensitized and challenged with HDM (pooled from three independent experiments). (G) Graphs of the number of alveolar macrophages (Siglec-F+CD11c+), T cells (Siglec-FCD11cCD3+), and neutrophils (Siglec-FCD11cGr-1+). (H) Representative periodic acid–Schiff stain of lung sections from naïve PBS-treated or HDM-sensitized and HDM-challenged mice (n = 3 in each HDM group; goblet cells in dark purple are seen lining the airways in HDM-treated Pparg+ mice). Scale bar, 250 μM. Mann-Whitney U test was used for all comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

To address TH2 cell function directly, we analyzed the CD4+ T cell response to HDM in the lung and lung-draining mediastinal lymph node (medLN). Comparable numbers of total cells and CD4+CD44+ memory cells were present in the lung and medLN of PpargLckΔ and control Pparg+ mice after sensitization and challenge with HDM (Fig. 3, A to C). However, significantly fewer memory CD4 T cells in the lung produced TH2 cytokines, including IL-5 and IL-13 (IL-5+IL-13, P = 0.0006; IL-5+IL-13+, P = 0.002) (Fig. 3, D and E). The frequencies of IL-4+ and IL-17+ memory CD4 T cells in the lungs of mice were similar between PpargLckΔ and control Pparg+ mice (fig. S1). In the medLN, the frequency of memory CD4 T cells producing IL-5 and IL-13 in the medLN was similar between PpargLckΔ and control Pparg+ mice (Fig. 3F), suggesting that PPAR-γ specifically promoted the pathogenic functions of TH2 cells in the effector tissue and was not important for the initial priming of this population. Thus, PPAR-γ was important for the differentiation of IL-5– and IL-13–secreting TH2 cells but was not required for IL-4 production by CD4 T cells.

Fig. 3 PpargLckΔ mice fail to develop robust TH2 cell responses.

(A) Representative plots of CD4 versus CD44 expression on lymphocytes from the lungs of WT control Pparg+ and PpargLckΔ mice sensitized and challenged with HDM on day 15. (B and C) Total cell number and CD4+CD44+ cell number in the lung (B) and the medLN (C) in control Pparg+ and PpargLckΔ mice after HDM sensitization and challenge. (D) Representative plots of IL-13 versus IL-5 expression from gated lung CD4+CD44+ cells in control Pparg+ and PpargLckΔ mice after restimulation for 3 hours with PMA and ionomycin. (E and F) Frequency of cytokine-producing cells in the lung (E) and medLN (F) (each symbol is an individual mouse) (PpargLckΔ, n = 10; Pparg+, n = 11, pooled from two independent experiments). N.S., not significant. Mann-Whitney U test was used for all comparisons. **P < 0.01 and ***P < 0.001.

PPAR-γ expression promotes protective TH2 cell–mediated immune responses to H. polygyrus

To determine whether PPAR-γ played a similar role in other type 2 immune responses, we infected PpargLckΔ and control Pparg+ mice with H. polygyrus and analyzed the response. The total number of lymphocytes and memory CD4 T cells in the mesLN was greatly increased in infected compared with noninfected mice but was not significantly reduced in worm-infected PpargLckΔ mice 15 days after infection (Fig. 4A). Furthermore, the proportion of memory CD4 T cells secreting both IL-5 and IL-13 was significantly reduced in the mesLN of worm-infected PpargLckΔ mice (P = 0.0279) (Fig. 4B), as was the frequency of eosinophils (P = 0.0004) (Fig. 4C and fig. S2). Similar to the HDM model, normal frequencies of CD4 T cells expressed the cytokines IL-4 and IL-17 (Fig. 4, D and E, and fig. S2). This suggested that PPAR-γ was required for the production of IL-5 and IL-13 but not for the production of IL-4 by TH2 cells. After primary infection with H. polygyrus, levels of total IgE in the serum of control Pparg+ mice were significantly higher than those in PpargLckΔ mice (Fig. 4F). Although robust type 2 responses to H. polygyrus were observed in Pparg+ mice, they showed no enhanced ability to clear the parasite 15 days after primary infection (Fig. 4G), in line with the persistent nature of this parasite after primary infection (29). To test whether T cell–derived PPAR-γ conferred protective immunity to H. polygyrus, we dewormed mice 2 weeks after primary infection and reinfected them 1 week later. Upon reinfection, PpargLckΔ mice had a greatly impaired ability to control worm load (P = 0.0051) (Fig. 4G). Hence, PPAR-γ expression in T cells is important for TH2 cell responses to H. polygyrus and promotes protective immunity to this pathogen.

Fig. 4 PPAR-γ promotes type 2 immunity to H. polygyrus.

Mice were infected with H. polygyrus (Hp), and responses in the mesLN and Peyer’s patches were measured by flow cytometry 15 days after infection. (A) Total and CD4+CD44+ cell number in mesLN in littermate control Pparg+ (infected, n = 12; uninfected, n = 6) and PpargLckΔ mice (infected, n = 11; uninfected, n = 4) 15 days after H. polygyrus infection (pooled from two to four independent experiments). (B) The frequency of IL-5+IL-13+ cells among gated CD4+CD44+ cells in mesLN and Peyer’s patches of control Pparg+ and PpargLckΔ mice of infected and uninfected mice. (C) Graph of eosinophil (Siglec-F+CD11c) frequency 15 days after infection. (D and E) The frequency of IL-4+ and IL-17+ cells among gated CD4+CD44+ cells in mesLN and Peyer’s patches of control Pparg+ and PpargLckΔ mice of infected and uninfected mice. (F) Serum concentration of total IgE in mice 15 days after infection, as measured by enzyme-linked immunosorbent assay (ELISA). One representative of two experiments is shown (Pparg+ Hp, n = 6; PpargLckΔ Hp, n = 4; uninfected, n = 3). (G) Viable adult worms per gut 15 days after primary infection with H. polygyrus (n = 6 to 8 mice pooled from two independent experiments) and 15 days after secondary infection (n = 10 to 11 mice pooled from two independent experiments). Mann-Whitney U test was used for all comparisons. *P < 0.05 and **P < 0.01.

Agonists of PPAR-γ induce ST2 expression on TH2 cells in vitro

To investigate the role of PPAR-γ on TH2 cell effector functions, we tested the impact of known PPAR-γ agonists in TH2 cell differentiation cultures. One such agonist is the prostaglandin derivative 15dΔ12,14-PGJ2 (15d-PGJ2) (30, 31). 15d-PGJ2 is derived from PGD2, which has been postulated to potentiate inflammation during asthma, partly by stimulating the receptor CRTH2 (32). The addition of 15d-PGJ2 did little to directly promote IL-5 or IL-13 production in TH2 cell differentiation cultures (Fig. 5A). However, 15d-PGJ2 notably up-regulated transcription of the ST2 gene (Il1rl1) (Fig. 5B). This was intriguing because a recognized caveat of classical TH2 cell differentiation assays is the inefficient induction of ST2 on the surface of TH2 cells (33). The addition of 15d-PGJ2 to TH2 cell differentiation cultures induced ST2 expression on about 10% of differentiating TH2 cells after 7 days of culture (P < 0.01) (Fig. 5, C and D). The synthetic PPAR-γ agonist pioglitazone (PIO) induced a similar increase in ST2 expression in differentiating TH2 cells (P < 0.05) (Fig. 5E), and the activity of 15d-PGJ2 and PIO appeared to be PPAR-γ–dependent (15d-PGJ2, P = 0.0002; PIO, P = 0.011) (Fig. 5F). These results imply that addition of PPAR-γ ligands to TH2 cell differentiation cultures may more accurately recapitulate physiological TH2 cell differentiation, by inducing IL-33R expression for instance.

Fig. 5 Agonists of PPAR-γ promote ST2 expression in TH2 cell differentiation cultures.

Naïve CD4 T cells were cultured under TH2 cell differentiation conditions in the presence or absence of 15d-PGJ2 for 7 days. (A) Cytokine expression was analyzed by intracellular staining (ICS). Representative plots of IL-13 versus IL-5 expression. (B) Il13 and Il1rl1 mRNA expression was quantified after 7 days of culture in the presence or absence of 15d-PGJ2 (n = 6 to 7 separate wells pooled and normalized to the level in TH2-only cultures). (C) Expression of ST2 versus fluorescence parameter 1 (FL-1) in representative plots. (D and E) Graphical representation of ST2 expression in the presence of the indicated doses of 15d-PGJ2 (D) or PIO (E) over time (n = 6 to 8 separate wells over two to three independent experiments). (F) In separate experiments, naïve CD4 T cells from Pparg+ and PpargLckΔ mice were cultured under TH2 cell differentiation conditions in the presence of 15d-PGJ2 or PIO, and the frequency of ST2+ cells was quantified after 7 days (n = 7 to 11 separate wells over three independent experiments). (D) and (E) were analyzed using one-way ANOVA and Bonferonni’s test for multiple comparisons to TH2 cells in the absence of compounds. Mann-Whitney U test was used in (B) and (F). *P < 0.05, **P < 0.01, and ***P < 0.001.

CD4 T cells from PpargLckΔ mice express reduced levels of the IL-33R subunit ST2

To validate our findings from in vitro cell cultures, we analyzed the expression of ST2 on CD4 T cells in HDM-induced airway inflammation and after nematode infection. In the lungs of PpargLckΔ mice administered HDM, the frequency of memory CD4 T cells expressing ST2 was considerably reduced (P = 0.0002) (Fig. 6, A and C), as was the mean fluorescence intensity of ST2 expression on ST2+ memory CD4 T cells (P < 0.0001) (Fig. 6E). These differences were not observed in the medLN (Fig. 6, B and D), reinforcing the notion that PPAR-γ activation is important for the promotion of pathogenic TH2 cells in the lung tissue directly (Fig. 3). Similarly, the frequency of ST2+ cells (P = 0.0007) and the intensity of ST2 expression (P = 0.0127) on memory CD4 T cells [CD4+CD44+Foxp3 cells to exclude gut-associated Tregs (34)] were also reduced in the mesLN and Peyer’s patches of PpargLckΔ mice in response to H. polygyrus infection (Fig. 6, F to I).

Fig. 6 PPAR-γ promotes the expression of ST2 in TH2 cells in vivo.

(A to E) Control Pparg+ and PpargLckΔ mice sensitized and challenged with HDM were analyzed for ST2 expression in medLN and the lung. (A) Representative plots of CD4 versus ST2 expression on gated lung CD4+CD44+ cells. (B to E) Graphs of the frequency of ST2+ cells among memory CD4+ cells in the medLN (B), the lung (C), and the MFI of ST2 expression on ST2+ memory CD4+ cells (D and E). (F to I) Control Pparg+ mice and PpargLckΔ mice were infected with H. polygyrus, and ST2 expression on CD4 T cells was analyzed by flow cytometry 15 days after infection. Graphs of the frequency of ST2+ cells among CD4+CD44+Foxp3 cells (F and G) and MFI of ST2 on ST2+CD4+CD44+Foxp3 cells (H and I) in the mesLN and Peyer’s patches 15 days after infection with H. polygyrus (n = 6 to 8 mice pooled from two independent experiments). (J) ST2+CD44+CD4+ cells from the lungs of HDM-sensitized and HDM-challenged Pparg+ and PpargLckΔ mice were purified and analyzed for genome-wide mRNA expression. Heat map showing the 29 genes that were consistently up-regulated or down-regulated between groups. Values are log2-transformed. (K to M) Galnt3, Plin2, and Ltb4r1 mRNA expression was quantified after 7 days of culture in the presence or absence of 15d-PGJ2 (n = 5 to 7 separate wells pooled and normalized to the level in TH2-only cultures). Mann-Whitney U test was used for all comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

To verify that PPAR-γ was required in a cell-intrinsic fashion for pathogenic TH2 cell differentiation and that the effects observed were not due to alterations in non-CD4 T cells or an altered cytokine milieu, we created 1:1 chimeras of WT and PpargLckΔ cells. Congenic CD45.1+ mice were lethally irradiated and infused with bone marrow cells from WT (CD45.1+CD45.2+) and PpargLckΔ (CD45.2+ only) mice (fig. S3A). This experimental setup allowed us to differentiate between transferred WT, PpargLckΔ, and radioresistant host cells. After 8 weeks, chimeric mice were sensitized and challenged with HDM, and the TH2 cell response in the lung was analyzed. Very few memory CD4 T cells from the PpargLckΔ fraction produced both IL-5 and IL-13 compared with the WT cell fraction (P = 0.0156) (fig. S3B). Similar to the results in PpargLckΔ mice, the frequency and intensity of ST2 expression in memory CD4 T cells from the PpargLckΔ fraction were severely impaired compared with WT cells in the same mouse (fig. S3C). Furthermore, in control experiments to test for effects of the Lck-Cre transgene alone, HDM was administered to Lck-Cre+Pparg+/+ and Lck-CrePparg+/+ littermate control mice. No difference in airway eosinophilia, frequency of ST2+ cells, or mean fluorescence intensity (MFI) of ST2 expression in the lung was observed (fig. S4). Together, this demonstrates that PPAR-γ instructs TH2 cell differentiation and the expression of ST2 in a cell-intrinsic manner and, furthermore, that PPAR-γ signals are crucial for the full up-regulation of ST2 on CD4 T cells in the lung.

Whole-genome expression arrays pinpoint genes affected by loss of PPAR-γ

To gain more mechanistic insight into what genes PPAR-γ may regulate, we performed whole-genome arrays of CD4+CD44+ST2+ cells from the lungs of mice sensitized and challenged with HDM. Expression arrays showed no difference in the expression of master transcriptional regulators, including Foxp3, Gata3, Bcl6, Tbx21, and Rorc, between cells sorted from control Pparg+ and PpargLckΔ mice (fig. S5). A total of 29 annotated genes appeared to be differentially expressed by at least twofold between CD4+CD44+ST2+ cells from Pparg+ and PpargLckΔ mice (Fig. 6J). These included a number of known TH2 cell and asthma risk factors such as Il5, Il13, and Ltb4r1, as well as PPAR-γ target genes such as Chi3l3. The expression of several factors involved in carbohydrate synthesis, metabolite transport, or lipid storage, such as Galnt3, Tgtp2, Slc7a8, Slc37a2, and Plin2, was increased in PPAR-γ–proficient cells, whereas two small GTPases, Tagap1 and Rras2, were reduced. To investigate whether the expression of some of these targets could be regulated by PPAR-γ, we analyzed the expression of Galnt3, Plin2, and Ltb4r1 in TH2 cells cultured in the presence or absence of 15d-PGJ2. We observed notable increases in the expression of Galnt3 and Plin2 (P = 0.0025) (Fig. 6, K and L) after the addition of 15d-PGJ2 to culture, whereas Ltb4r1 mRNA showed no significant trend (Fig. 6M). These results suggest that although PPAR-γ may not directly induce the production of effector molecules, it may promote the expression of a key set of metabolic regulators that support TH2 cell functions in the lung.

PPAR-γ supports a population of resident TH2 cells and ST2+ Tregs in VAT

The notable impact of PPAR-γ on ST2 expression in CD4 T cells prompted us to investigate VAT, which contains prominent populations of CD4 T cells and ILC2 that have been implicated in the regulation of glucose sensitivity (19, 35). Pparg mRNA was detectable in both CD4 T cells and ILC2 from VAT of Pparg+ mice (fig. S6A). Note that expression of Pparg mRNA was higher in purified macrophages from the VAT than in ILC2 or CD4 T cells. Likewise, in vitro–generated M2 macrophages expressed about eightfold more Pparg mRNA than differentiating TH2 cells (fig. S6B). It is possible that the lower levels of Pparg mRNA in TH2 cells reflect the fact that only a small proportion of differentiating TH2 cells express this factor. This would explain why ST2 is only up-regulated in around 10% of cells in vitro after stimulation with 15d-PGJ2. The lack of reliable antibodies for intracellular detection of PPAR-γ at the single-cell level makes this a difficult premise to test.

In PpargLckΔ mice, Pparg mRNA was detected at normal levels in purified ILC2 and macrophages, whereas it was completely absent from purified CD4 T cells (fig. S6A). Thus, Pparg was deleted from T cells but not from ILC2, despite reports of Lck expression in ILC2 (36). In line with their normal expression of Pparg, ILC2 were present at standard frequencies in VAT of PpargLckΔ mice (fig. S6, C and D). Furthermore, the frequency of ILC2 in response to H. polygyrus and HDM was, for the most part, comparable between Pparg+ and PpargLckΔ mice (fig. S7), despite a moderate increase in the total frequency of ILC2 in the lungs of PpargLckΔ mice after HDM administration. In a short-term model of ILC2-mediated airway inflammation with recombinant (r) IL-33, airway eosinophilia and ILC2 responses were induced comparably in Pparg+ and PpargLckΔ mice (fig. S8). Hence, ILC2 responses were not greatly affected by the absence of PPAR-γ in T cells.

Because VAT CD4 T cells lacked Pparg mRNA, we further analyzed this population. Previous studies have shown that VAT Tregs express high levels of ST2 (19, 35). In VAT of PpargLckΔ mice, we observed a notable reduction specifically in ST2-expressing CD4 T cells both within the Foxp3+ Treg compartment (P = 0.0051) and among conventional CD4+Foxp3 cells (P = 0.0039) (Fig. 7, A and B). This reduction was not observed in other organs analyzed, including the thymus, spleen, medLN, lung, and colon (fig. S9), where ST2+ Tregs have been suggested to regulate tolerance to the gut microbiota (34). In response to HDM, the overall numbers of Tregs were comparable between Pparg+ and PpargLckΔ mice (fig. S10, A and B); however, a specific reduction in the frequency of ST2-expressing Tregs was observed in PpargLckΔ mice (P = 0.0159) (fig. S10B). No such difference was observed in the H. polygyrus model (fig. S10C). Thus, ST2+ Tregs in VAT are dependent on PPAR-γ, and responses of this population to the HDM allergen in the lung are also somewhat dependent on PPAR-γ.

Fig. 7 PPAR-γ supports resident TH2 cells in the VAT.

Populations of CD4 T cells in VAT of 12- to 20-week-old mice were analyzed by flow cytometry (n = 9 mice per group pooled from three experiments). (A) Representative dot plots of Foxp3 versus ST2 expression on gated CD4 T cells from VAT of control Pparg+ and PpargLckΔ mice. (B) Quantification of ST2+Foxp3, ST2+Foxp3+, and ST2Foxp3+ CD4+ cells from VAT of PpargLckΔ mice. (C) Representative plots of IL-5 versus IFN-γ expression on gated CD4 T cells from VAT of control Pparg+ and PpargLckΔ mice. (D) Quantification of IL-5 and IFN-γ single-positive (SP) CD4+ cells. (E) Weight of VAT from 20-week-old mice on a normal chow diet. (F) Glucose concentration in plasma after injection of glucose over 120 min (n = 7 to 10 mice pooled from two independent experiments). For statistical analysis, area under the curve was calculated. Mann-Whitney U test was used for all comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

To test the cytokine secretion potential of VAT CD4 T cells, lymphocytes were restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin. The frequency of CD4 T cells that produced IL-5 in VAT of PpargLckΔ mice was significantly reduced compared with that in control mice (P = 0.0056), although there was a reciprocal increase in cells expressing interferon-γ (IFN-γ) (P = 0.0314) (Fig. 7, C and D). This suggests that PPAR-γ promotes a resident population of TH2 cells in VAT, in addition to ST2+ Tregs.

Type 2 cytokines, such as IL-4 and IL-5, and Treg-derived IL-10 have been classically viewed to mediate beneficial metabolic effects, for instance, by promoting sensitivity to glucose (19, 37). However, we observed that aged PpargLckΔ mice were leaner (Fig. 7E) and exhibited significantly greater control of plasma glucose levels after overnight fasting (P = 0.0051) (Fig. 7F), suggesting that ST2+ Tregs and TH2 cells may impair glucose sensitivity in the context of aging.

Together, our study identifies PPAR-γ as an important factor for the full effector differentiation of ST2+ IL-5– and IL-13–secreting TH2 cells, as well as for responses of ST2+ Tregs in VAT and in response to the allergen HDM.

DISCUSSION

PPAR-γ is typically regarded as an immunosuppressive factor. It serves to impair T cell priming by dendritic cells (38), suppress inflammatory chemokine and cytokine production by epithelial cells of the gut or airways (16, 17), promote the conversion of naïve CD4 T cells to Tregs (39, 40), and maintain the Treg population in VAT (19). Contrary to these well-described anti-inflammatory functions, we have demonstrated here that PPAR-γ has an important function in TH2 cells, where it is critical for TH2 cell–mediated allergic inflammation and immunity to worm infection. We found little evidence that PPAR-γ was required for IL-4 production by TH2 cells and rather supported a population of IL-5+IL-13+ TH2 cells, considered a pathogenic subset of cells, for instance, in the context of airway inflammation. In light of a recent study proposing that pathogenic TH2 cells complete their differentiation in the lung tissue directly and depend on tissue-specific signals such as IL-33 (14), our results suggest that PPAR-γ agonists up-regulate the expression of ST2 on differentiating TH2 cells in the lung and thereby promote their potential for IL-5 and IL-13 secretion. Thus, strategies to specifically target PPAR-γ in IL-5+IL-13+ TH2 cells may effectively ameliorate airway inflammation.

Supportive of our observations in TH2 cells, PPAR-γ was previously shown to promote M2 macrophage differentiation and to be induced by IL-4 receptor signaling and STAT-6 activation (18, 41). In dendritic cells and macrophages, PPAR-γ and STAT-6 have been shown to orchestrate a joint transcriptional program (42). This has made the association between PPAR-γ and the TH2 cell lineage intuitive, yet it has remained unstudied to date. PPAR-γ may perform similar functions in TH2 cells, for instance, by facilitating the binding of STAT-6 and/or GATA-3 to certain TH2 gene loci and regulating changes in chromatin architecture. However, previous studies and our own results do not suggest that PPAR-γ is a strong inducer of effector TH2 cytokine gene transcription. It is more likely that PPAR-γ regulates various “nonclassical” TH2 cell target genes such as Arg1 and Chi3l3. Furthermore, given the known interplay of PPAR-γ in lipid metabolism, it is enticing to hypothesize that PPAR-γ facilitates the requirements of TH2 cell metabolism, for instance, by enhancing the expression of genes including Galnt3 and Plin2.

One limitation of our study was an inability to accurately detect PPAR-γ protein. This has various implications. First, whether all TH2 cells or only a subset of “pathogenic” TH2 cells express PPAR-γ is still unclear. Gene expression analysis using single-cell RNA-sequencing approaches or following purification of pathogenic TH2 cells using an IL-5 reporter strain may help clarify this point. Second, we were unable to reliably precipitate PPAR-γ from DNA and identify loci to which it had bound. Nonetheless, in adipocyte differentiation cultures, super-enhancers associated with PPAR-γ–responsive elements have been found at many loci, including an enhancer of Il1rl1 (43). Thus, although we were unable to directly confirm the binding of PPAR-γ to this locus in TH2 cells, the induction of ST2 protein after agonism of PPAR-γ suggests that this may be one transcriptional target.

Although we have demonstrated here a clear amplifying function for PPAR-γ in TH2 cell–mediated responses, preclinical studies have repeatedly demonstrated that TZDs ameliorate airway inflammation and improve respiratory function in animal models. This effect has often been linked to inhibition of the inflammatory activity of airway epithelial cells or the priming function of dendritic cells (21, 22). The therapeutic effects of TZDs in small animal models even led to trials in asthmatic patients; however, these showed that agonists of PPAR-γ had limited benefits (23, 44). Our data suggest that activation of PPAR-γ may in fact exacerbate type 2–mediated airway inflammation in some cases. Although TZD use in patients with type 2 diabetes mellitus has proven useful, side effects of TZDs include edema, weight gain, and heart failure, which all testify to a potentially inflammatory function for PPAR-γ (20, 45). Note that SNPs in IL1RL1 and IL33 are strongly associated with heart disease and asthma (12, 13), and it is possible that the side effects of TZDs on cardiac function may be linked to the activation of this highly inflammatory ST2–IL-33 axis.

A recent area of contention has revolved around the impact of PPAR-γ agonists on cell populations in VAT. Although TZDs have been shown to improve glucose sensitivity (20), whether Tregs were the critical mediators of this effect or whether other cell populations in VAT were responsible was unclear (19, 46). We have shown here that PPAR-γ is expressed by both CD4 T cells and ILC2 and, furthermore, that this expression is important for the accumulation of TH2 cells. Because both ILC2 and CD4 T cells in VAT have previously been shown to regulate metabolism, our results suggest that TZDs may also act on ILC2 and TH2 cells to regulate glucose and insulin sensitivity. Recent single-cell RNA-sequencing analysis of human ILC populations also highlighted the fact that PPAR-γ was enriched in ILC2 from human tonsils, suggesting that PPAR-γ expression is a trait conserved not only between adaptive and innate type 2 lymphocytes but also across species (47). Another recent observation in the study by Bapat et al. (46) was that Treg cells that accumulated with age impaired glucose sensitivity. This was odd because Tregs have typically been thought to suppress inflammation and promote glucose sensitivity (19, 48), especially in the context of obesity. Our results support their assertion that PPAR-γ–dependent accumulation of Tregs (and, potentially, TH2 cells as well) may act to impair glucose sensitivity, in the setting of aging. Hence, TZDs are likely to target Tregs, TH2 cells, and ILC2 cells in VAT, but their therapeutic effects in the context of either aging or obesity may be disparate.

Last, in the H. polygyrus model of infection, we observed reduced levels of IL-5+IL-13+ TH2 cells and severely reduced serum IgE responses, despite normal IL-4 production by CD4 T cells. It is possible that the reduction in IgE reflects the reduction in CD4 T cell–derived IL-13, although we cannot rule out the possibility that IL-4 was reduced at earlier time points and that this affected IgE levels on day 15. Although IL-5, eosinophils, and IgE play a limited role in expulsion or control of H. polygyrus (29), it is clear that loss of PPAR-γ in T cells impaired immunity to secondary infection with this pathogen. The impact of PPAR-γ in other models of helminth infection where IL-5 and eosinophils can play a greater role is worth exploring (49, 50).

MATERIALS AND METHODS

Study design

The aim of this study was to characterize the role of PPAR-γ in type 2 immunity. To achieve this aim, we analyzed cellular, histological, and molecular components from age-matched littermate control WT and gene-targeted mice. Cages were randomly assigned to uninfected, infected, PBS-treated, or allergen-treated groups. Mice within one cage were always treated in the same fashion to avoid the risk of transmitting pathogens or allergens from exposed to unexposed mice. Power calculations were performed on the basis of many previous studies that have been conducted by our groups in the context of allergen challenge and helminth infection. The assessment of worm burden and histological sections was performed in a blinded fashion. All experiments were conducted in accordance with the Stockholms Norra djurförsöksetiska nämnd.

Mice and generation of bone marrow chimeras

WT C57BL/6, Lck-Cre+, Ppargfl/fl, and C57BL/6.CD45.1 congenic mice were bred and maintained at the Department of Microbiology, Tumor and Cell Biology, Karolinska Institute. Gata3+/+ and Gata3+/− mouse spleens were transported overnight from Erasmus MC (Rotterdam, Netherlands) to Karolinska Institute (Stockholm, Sweden) for further analysis. For generation of bone marrow chimeras, C57BL/6 CD45.1 congenic mice were lethally irradiated with 2× 5-Gy γ-irradiation 3 hours apart. The following day, mice were reconstituted intravenously with a 1:1 mixture of CD45.1+CD45.2+ WT and CD45.1CD45.2+ PpargLckΔ bone marrow cells. Mice were kept on antibiotics for 3 weeks and used in experiments 8 weeks after irradiation.

Models of asthma and H. polygyrus infection

House dust mite

Four- to 5-week-old mice were anesthetized briefly with isoflurane. For intranasal sensitization, mice received 1 μg of HDM in 40 μl of PBS. One week after sensitization, mice were challenged for 5 consecutive days with 10 μg of HDM administered intranasally under light anesthesia with isoflurane. Four days after the last administration, mice were sacrificed, and their organs were dissected for analysis. BAL was performed by two consecutive flushes of the lung with 1 ml of PBS. The medLN and spleen were dissected through 100 μM sieves and analyzed by flow cytometry. Periodic acid–Schiff staining was performed at the Morphological Phenotype Analysis facility at Karolinska Institute.

Heligmosomoides polygyrus

Three- to 4-week-old mice were infected with 200 L3 (third-stage H. polygyrus larvae), and euthanized 2 weeks after infection. For removal of worms, the mice were treated with pyrantel pamoate (1 mg/g; Fyrantel vet) for 3 consecutive days, starting 2 weeks after infection. After anti-helminth treatment, mice were screened for eggs in feces. No eggs were shed when reinfection with 200 L3 was given 3 days after the last treatment with pyrantel pamoate. Mice were euthanized 2 weeks after reinfection.

TH cell differentiation assays

For TH cell differentiation, the spleen was harvested, and naïve CD4+ T cells were enriched with the Naïve CD4+ T Cell Isolation Kit (Miltenyi Biotec) or purified on the basis of CD4, CD44, and CD25 using a FACSAria Fusion (BD). All cell culture was performed in Iscove’s modified Dulbecco’s medium (IMDM) or RPMI supplemented with penicillin/streptomycin, glutamine, 2-mercaptoethanol (all from Invitrogen), and 8% heat-inactivated fetal calf serum (FCS) (Sigma). For all differentiation conditions, 1 × 105 enriched naïve CD4+ T cells in 200 μl of culture medium were plated in 96-well polystyrene round-bottom plate precoated with anti-CD3 (4 μg/ml; 145-2C11, BD) and soluble anti-CD28 (1 μg/ml; clone 37.51, BD). For TH1 cell differentiation, the culture was conditioned in rmIL-12 (100ng/ml; PeproTech) and anti–IL-4 (5 μg/ml; 11B11, BD); to induce TH2, we added rmIL-4 (20ng/ml; Sigma) and anti–IFN-γ (10 μg/ml; XMG1.2, Bio X Cell) to the medium; TH17 cells were differentiated in rhTGF-β (5 ng/ml; R&D), rmIL-6 (20 ng/ml; BD), and anti–IFN-γ (10 μg/ml; XMG1.2, Bio X Cell); Tregs were induced in rhTGF-β (5 ng/ml; R&D) and anti–IFN-γ (10 μg/ml; XMG1.2, Bio X Cell).

For PIO or 15d-PGJ2 (Sigma)–treated TH2 cultures, various doses of the drugs were added to the culture on day 0, and cells were harvested for ST2 expression between days 4 and 7. For cultures that were maintained for 7 days, culture medium was replenished on days 4 and 6 with the components of TH2 differentiation at appropriate concentrations, as described above.

Restimulation (ICS, ELISA, and PMA)

Cell culture was performed in either RPMI-1640 or IMDM supplemented with penicillin/streptomycin, glutamine, 2-mercaptoethanol (all from Invitrogen), and 8% heat-inactivated FCS (Sigma). To analyze cytokine production, we added PMA (50 ng/ml) and 5 μM ionomycin (both from Sigma) to cultures of total medLN, mesLN, VAT, Peyer’s patches, or lung for 2.5 to 3 hours in the presence of Brefeldin A (Sigma). The BD Fixation/Permeabilization Kit was used for intracellular staining of cells for cytokines. The eBioscience Fixation Kit was used when intranuclear staining of Foxp3 was performed.

Quantitative PCR and array

RNA was obtained using the TRIzol isolation reagent (Thermo Scientific) and isolated according to the manufacturer’s instructions. The RNA was reverse-transcribed using Invitrogen SuperScript (generations 3 and 4), and samples were analyzed by SYBR Green–based reverse transcription PCR on the Bio-Rad CFX384 machine.

For the microarrays in Fig. 1, CD4+ T cells (CD4+CD45+F4/80) from the lung were purified from naïve PBS-treated or HDM-treated mice. Samples were analyzed on a GeneChip Mouse Gene 2.0 ST Array (Affymetrix). Data were deposited at the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database (GSE72005). For the microarrays in Fig. 6, ST2+ memory CD4 T cells were purified, and samples were analyzed on a GeneChip Mouse Gene 2.1 ST Array (Affymetrix). Only 29 annotated genes (31 probes) were consistently differentially expressed with at least a twofold difference between PpargLckΔ and control Pparg+ mice. T cell receptor, immunoglobulin, and unannotated genes were discarded from the analysis at this time. A full description is provided in the Supplementary Materials.

Statistical analysis

A nonparametric Mann-Whitney U test was used to compare two groups. One-way analysis of variance (ANOVA) and Bonferonni’s post hoc test were used for multiple comparisons. For glucose tolerance testing, statistical significance was analyzed by Mann-Whitney U test after measuring the area under the curve in both groups of mice. Mean and SEM are shown in all graphs. *P < 0.05, **P < 0.01, and ***P < 0.001.

SUPPLEMENTARY MATERIALS

immunology.sciencemag.org/cgi/content/full/2/9/eaal5196/DC1

Materials and Methods

Fig. S1. Normal expression of IL-4 and IL-17 by lung memory CD4 T cells after HDM sensitization and challenge.

Fig. S2. Representative profiles of mesLN cells after H. polygyrus and graphs of cytokine production in Peyer’s patches.

Fig. S3. The effects of PPAR-γ on IL-5, IL-13, and ST2 expression are cell-intrinsic.

Fig. S4. Normal BAL eosinophilia and ST2 expression after HDM administration into control Lck-Cre+ WT mice.

Fig. S5. Sort strategy for whole-genome expression arrays.

Fig. S6. ILC2 in VAT express PPAR-γ but are not targeted for Pparg gene deletion in PpargLckΔ mice.

Fig. S7. The effect of PPAR-γ on ILC2 frequency after HDM administration and H. polygyrus infection.

Fig. S8. Normal ILC2 responses in PpargLckΔ mice.

Fig. S9. Normal frequencies of ST2+ Tregs at baseline in various organs.

Fig. S10. Analyzing the requirement for PPAR-γ in Tregs in the HDM and H. polygyrus model.

Raw data (Excel)

Array data (Excel)

REFERENCES AND NOTES

Acknowledgments: We thank L. Westerberg and M. Wahlgren for providing important tools, B. W. S. Li for technical support, and G. Arulampalam for useful discussions. Histology was performed at the Core Facility for Morphological Analysis, Laboratory Medicine, Karolinska Institute, Sweden. Funding: J.M.C. was supported by a Swedish Research Council Young Investigator Grant and grants from the Swedish Cancer Society and the Åke Wiberg Foundation. Author contributions: T.C., C.A.T., X.F., J.M.S., L. Rohrbeck, B.J.C., S.K.S., S.N., L. Rausch, and J.M.C. performed experiments. G.B.K.H., B.N.L., S.N., M.C.I.K., and R.W.H. provided important reagents. J.M.C. conceived the study. J.M.C. and T.C. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Data for Fig. 1 were deposited at the NCBI GEO database (GSE72005).
View Abstract

Navigate This Article