Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Apr 1.
Published in final edited form as: Am J Transplant. 2016 Feb 4;16(4):1173–1182. doi: 10.1111/ajt.13595

B Cell Activating Transcription Factor (BATF) plays a critical role in the pathogenesis of anti-MHC induced Obliterative Airway Disease

Z Xu 1, S Ramachandran 1, M Gunasekaran 1, D Nayak 1, N Benshoff 1, R Hachem 3, A Gelman 1,2, T Mohanakumar 1,2,*
PMCID: PMC4803590  NIHMSID: NIHMS758191  PMID: 26844425

Abstract

Antibodies (Abs) against MHC results in T helper-17 (Th17) mediated immunity against lung self-antigens (SAgs), K-α1 tubulin and collagen V and Obliterative Airway Disease (OAD). Since B cell activating transcription factor (BATF) controls Th17 and autoimmunity, we proposed that BATF may play a critical role in OAD. Anti-H2Kb was administered intrabronchially into Batf−/− and C57BL/6 mice. Histopathology of the lungs on days 30 and 45 following Abs administration to Batf−/− mice resulted in decreased cellular infiltration, epithelial metaplasia, fibrosis and obstruction. There was lack of Abs to SAgs, reduction of SAgs specific IL17 T cells, IL-6, IL-23, IL-17, IL-1β, FGF-6 and CXCL12 and decreased Janus kinase 2, STAT3, and Retinoid-related orphan receptor gamma-T. Further, miR-301a, a regulator of Th17, was reduced in Batf−/− mice in contrast to up regulation of miR-301a and down regulation of PIAS3 in anti-MHC induced OAD animals. We also demonstrate increase in miR-301a in the bronchoalveolar lavage cells from lung transplant (LTx) recipients with Abs to HLA. This was accompanied by reduction in PIAS3 mRNA. Therefore, we conclude that BATF plays a critical role in the immune responses to SAgs and pathogenesis of anti-MHC induced rejection. Targeting BATF should be considered for preventing chronic rejection following human LTx.

Introduction

Development of bronchiolitis obliterans syndrome (BOS) following human lung transplantation (LTx) significantly impedes long-term function of the allografts (1). Studies by us and others have shown that de novo development of donor specific antibodies (DSA) to mismatched HLA significantly increases the risk of development of BOS which often precedes the development of chronic lung allograft rejection (2, 3). However, the mechanisms by which antibodies (Abs) to HLA induces development of chronic rejection remain undefined. We developed a murine model in which intrabronchial administration of Abs to mouse MHC class I results in cellular infiltration, epithelial metaplasia, fibrosis of the native lungs, and development of obliterative airway disease (OAD) that pathologically resembles the lesions observed in BOS after human LTx (4). Administration of anti-MHC also results in the initiation of both cellular and humoral immune responses against lung associated self-antigens (SAgs), K-α1 tubulin (Kα1T) and Collagen V (ColV) (4). Blockade of IL-17 signaling by administration of anti-IL-17 Ab abrogated OAD lesions induced by anti-MHC indicating a crucial role for IL-17 in development of chronic rejection (4).

B cell activating transcription factor (BATF) is a basic leucine zipper transcription factor that dimerizes with the JUN proteins to direct patterns of activator protein-1 (AP1)-mediated gene expression in the immune system (5, 6). BATF is induced upon CD4+ T helper (Th) cell activation and is required exclusively for Th17 cells but not for Th1 or Th2 cell generation (7). Upon induction, BATF can bind to IL-17A and IL-17F intergenic regions and IL-21 and IL-22 promoter regions, and induce Th17 cytokine production by maintaining Retinoid-related orphan receptor alpha (RORα) and Retinoid-related orphan receptor gamma-T (RORγt) expression (7, 8). Mice lacking Batf failed to induce the Th17 transcription factor RORγt and failed to express Th17-specific cytokines such as IL-17A (7). Batf−/− mice were also resistant to experimental autoimmune encephalomyelitis (EAE), an autoimmune disease where Th17 cells play an indispensable pathogenic role (9). Furthermore, it has been demonstrated that Batf in B cells could control the expression of activation-induced cytidine deaminase and regulate switched Ab responses in vivo (10, 11). Therefore, we postulated that BATF mediated Th17 immune responses might be important for the development of immune responses to lung associated SAgs and pathogenesis of anti-MHC induced OAD. Our results demonstrated that Batf deficiency resulted in a significant reduction in cellular infiltration, epithelial metaplasia and fibrosis following intrabronchial administration of anti-MHC. Batf deficiency also resulted in significant reduction in miR-301a, a critical regulator of Th17 immune response and lack of Th17 responses and Abs to Kα1T and ColV.

Materials and Methods

Murine model of anti-MHC class I induced OAD

All experiments were performed in compliance with the guidelines of the Institutional Laboratory Animal Care and Use Committee of Washington University School of Medicine. B6.129S-Batftm1.1Kmm/J was obtained from Jackson Laboratory. Murine mAb to H2Kb (C57BL/6, 6~8 weeks, male, IgG2a), tested endotoxin free as measured by LAL assay was given into C57BL/6 or Batf−/− mice as described earlier (4). Anti-H2Kb (200 μg/dose) was administered into the lung on day 1, 2, 3, 6 and then weekly thereafter. C1.18.4 (isotype control) was administered as control. To determine the role of lymphocytes (T or B) in induction of autoimmunity in Batf−/− mice, 1×106 T or B cells isolated from splenocytes of C57BL/6 mice were injected intravenously on day-1 along with the first dose of anti-MHC intrabronchially.

LTx Recipients (LTxR)

Twenty-five LTxR were included in this study (Table S1). Among them, 15 developed DSA after LTx (DSA group) and the other 10 didn't develop DSA after transplantation. The mean fluorescent intensity values of DSA and Abs to MHC class I or II in the DSA group were included in Table S1. These 25 LTxR were matched for age, time from transplantation, underlying diagnosis, and immunosuppressive treatment. All LTxs were performed at Barnes Jewish Hospital/Washington University School of Medicine between 2011 and 2013. The study was approved by the institutional review board at the Washington University School of Medicine, and written informed consent was obtained from all patients enrolled for this analysis. Bronchoalveolar lavage (BAL) was collected during routine bronchoscopy, and cells isolated from BAL were stored at −80°C before RNA extraction.

Histological analysis

Lungs were fixed in 10% formaldehyde and sections cut at 5–10 μm thickness and stained with Hematoxylin & Eosin (H&E) and Masson's trichrome. Lesions that displayed cellular infiltration, epithelial abnormalities, and fibro-proliferation were subjected to morphometric analysis as previously described (4). Fibrosis was calculated using Optimas software version (Media Cybernetics, Rockville, MD), as a percentage of total area enclosed by basement membrane. Cellular infiltration and epithelial abnormalities were similarly calculated as a percentage of the total bronchiole and vessels visualized, respectively.

ELISPOT assay

Lung infiltrating lymphocytes were isolated using Ficoll-Paque density centrifugation as described previously (4). The kinetics of lung infiltrating cells, including neutrophils, macrophages, CD8+ T cells, CD4+ T cells, B cells, epithelial/endothelial cells was profiled previously (12). ELISPOT assays were performed as described previously (13). Briefly, 3×105 cells/well were cultured in triplicate in the presence of ColV (20 μg/ml) or Kα1T (1 μg/ml) and irradiated feeder autologous splenocytes (1:1 ratio) in cytokine Ab coated 96 well plates for 48–72h. The plates were developed according to the manufacturer's instruction and spots were analyzed in an ImmunoSpot Series I analyzer (Cellular Technology, Shaker Heights, OH), and the results were expressed as spots per million cells ± SE. Any spots obtained by culturing T-cell lines with antigen presenting cells alone without ColV or Kα1T were subtracted from number of spots in test cultures.

ELISA

ELISA plates were coated with purified ColV (BD Biosciences, San Jose, CA) or recombinant Kα1T (1 μg/ml) in PBS over night at 4°C. Serum samples were tested (1:250 and 1:500) and detection was done with anti-mouse IgG, IgM-HRP and developed using TMB substrate and read at 450 nm. Concentration of Abs was calculated based on a standard curve obtained with known concentrations of anti-ColV or anti-Kα1T (Santa Cruz Biotech) ran in the same plate.

Quantitative real-time PCR (qRT-PCR)

The mRNA expression levels of IL-6, IL-23, IL-17, IL-1β, FGF-6, CXCL-12, Janus kinase 2 (JAK2), STAT3, RORγt, and PIAS3 were quantified by qRT-PCR using gene-specific primers (Table S2). The data were normalized relative to GAPDH. Quantification of miR-301a was performed using TaqMan miRNA assay (Applied Biosystems). SnoRNA135 were used as reference control.

Statistical analysis

qRT-PCR results were compared by Mann Whitney U-test using GraphPad Prism program (Lo Jolla, CA). All data were expressed as mean ± SEM, unless otherwise specified. P value <0.05 was regarded as statistically significant.

Results

Batf deficiency leads to decreased cellular infiltration, endotheliitis, epithelial hyperplasia and fibrosis following MHC I treatment

In order to delineate an obligatory role for Batf in modulating OAD, we administrated anti-MHC to Batf−/− mice and normal BL/6 animals (WT) and analyzed on days 30 and 45 for the development of OAD. As shown in Figure 1, histological analysis by H&E staining of the lungs of Batf−/− mice recovered on day 30 following administration of anti-H2Kb demonstrated significant decrease in cellular infiltration around bronchioles (Figure 1A (c)) resulting in reduction of luminal occlusion (Figure 1A (c)) and epithelial metaplasia (Figure 1A (c)) compared to WT (Figure 1A (a)). Trichrome staining for collagen deposition also demonstrated a significant decrease of fibrosis in Batf−/− mice compared to WT (Figure 1A (g) and (e)). Significant reduction of lesions in anti-H2Kb treated Batf−/− mice on day 45 were also noted compared to WT (Figure 1B). Isotype Ab administered Batf−/− mice and WT animals did not demonstrate any lesion in the lung on days 30 and 45 (Figure 1A (b), (d) and Figure 1B (b), (d)). Morphometric analysis of H&E staining of the Batf−/− lungs recovered on day 30 following administration of anti-H2Kb demonstrated significant decreases in cellular infiltration around bronchioles (Batf−/−=18.2±1.8%; WT=54.1±8.6%, p<0.01, Figure 1C), resulting in marked reduction in epithelial metaplasia (Batf−/−=16.7±1.2%; WT=63.5±12.3%, p<0.01, Figure 1C). Trichrome staining for collagen deposition also demonstrated a significant decrease in fibrosis (Batf−/−=13.9±1.3%; WT=45.8±2.9%, p<0.01, Figure 1C) in Batf−/− animals. Taken together, these results demonstrated that Batf deficiency results in markedly decreased OAD lesions following administration of anti-MHC, suggesting a crucial role of Batf in OAD development.

Figure 1.

Figure 1

Open in a new tab

Batf deficiency leads to decreased cellular infiltration, endotheliitis, epithelial hyperplasia and fibrosis following administration of anti-MHC class I. Anti-H2Kb or control (C1.18.4) Ab was administered endobronchially in C57BL/6 mice (n=9) and Batf−/− mice (n=9) on days 1, 2, 3, and 6, and weekly thereafter. The lungs were harvested on day 15, 30 or 45 and analyzed by H&E and Trichrome staining. A representative picture from data obtained from five mice is presented in the figure. Original magnification, x100; Upper panels represent sections stained with H&E; Lower panels represent Trichrome staining. (A and B). Anti-H2Kb-treated mice on day 30 and 45. Lungs from the anti-H2Kb Ab-treated mice showed significant cellular infiltration around bronchia and vessel, and hyperplasia of the bronchial epithelium. A significant increase in the fibroproliferation, collagen deposition, and luminal occlusion was observed in the lungs of the anti-H2Kb Ab-treated mice. Mice treated with isotype control Ab or Batf−/− mice treated with anti-H2Kb Ab showed no significant morphological changes compared with the anti-H2Kb Ab-treated mice. (C). Morphometric analysis performed using NIS-Elements BR software of the cellular infiltration, epithelial metaplasia and collagen deposition. P-values were calculated with the Student's t-test; error bars represent SEM. Data are pooled from two independent experiments performed with three mice per group.

Batf deficiency results in decrease in the frequency of SAgs specific T lymphocytes secreting IFN-g and IL-17

In order to determine whether an immune responses to lung associated SAgs can be induced in Batf−/− mice following administration of anti-MHC, Kα1T and ColV-specific T lymphocytes that infiltrate the lungs at day 30 after anti-MHC administration were enumerated using ELISPOT. As shown in Figure 2A and 2B, anti-MHC-administered Batf−/− animals had a significant decrease in Kα1T and ColV-reactive T lymphocytes in comparison with WT. These SAg specific T cells include Kα1T-reactive T lymphocytes capable of secreting IFN-g (11.2±2.3 vs. 82.3±9.4, p<0.01), IL-17 (5.6±0.8 vs. 106.5±13.2, p<0.01) and IL-4 (10.9±2.1 vs. 41.3±7.8, p<0.01), and ColV-reactive cells secreting IFN-g (14.6±1.7 vs. 67.2±7.1, p<0.01), IL-17 (3.2±0.4 vs. 128.9±15.1, p<0.01) and IL-4 (7.9±0.5 vs. 34.8±5.5, p<0.01). Further, SAg specific T lymphocytes secreting IL-10 was significantly up regulated on day 30 both against Kα1T (58.6±9.2 vs. 37.8±6.3, p<0.01) and ColV (41.4±5.3 vs. 23.6±4.2, p<0.01) (Figure 2A and 2B). These results demonstrate that Batf is critical for the induction of immune responses against the SAgs, Kα1T and ColV upon administration of anti-MHC.

Figure 2.

Figure 2

Open in a new tab

Batf deficiency results in decrease in the frequency of SAg (Kα 1T and ColV) specific T lymphocytes secreting IFN-g and IL-17. (A). Frequency of the IFN-g, IL-17, IL-4, and IL-10-secreting T cells against Kα1T. (B). Frequency of the IFN-g, IL-17, IL-4, and IL-10-secreting T cells against ColV. Anti-H2Kb or control Ab was administered endobronchially in C57BL/6 (n=3) and Batf−/− mice (n=3) on days 1, 2, 3, and 6, and weekly thereafter. The lungs were harvested on day 30. T cells infiltrating the lungs were harvested by collagenase digestion, and the frequency of T cells secreting IFN-g, IL-17, IL-4, and IL-10 on stimulation with Kα 1T and ColV was analyzed by ELISPOT. The values are shown as means ± SEM using data obtained from two ELISPOT assays. P-values were calculated with the Student's t-test; error bars represent SEM. Mean results from two independent experiments are shown (n = 3/group).

Batf deficiency leads to decreased humoral immune responses against lung associated SAgs, Kα1T and ColV

De novo development of Abs against Kα1T and ColV following human LTx have been associated with development of chronic rejection (14) as well as in animal models of OAD induced by anti-MHC (4). Therefore, we analyzed serial serum samples following anti-MHC administration into Batf−/− mice for the development of Abs to Kα1T and ColV using ELISA. As shown in Figure 3, sera obtained on days 15 and 30 following administration of anti-MHC to Batf−/− mice had significantly reduced levels of Abs to Kα1T compared to WT mice (day 15: 17.5±2.3 vs. 87.5±7.6, p<0.01, and day 30: 85.7±5.3 vs. 223±13, p<0.01, Figure 3). In addition, sera obtained at 30 days after anti-MHC administration into Batf−/− mice also demonstrated significantly reduced levels of Abs to ColV compared to WT mice (97.8±6.9 vs. 144±11, p < 0.01, Figure 3). These results demonstrate that Batf also play an important role in production of Abs to lung associated SAgs following administration of anti-MHC.

Figure 3.

Figure 3

Open in a new tab

Batf deficiency leads to decreased humoral immune responses against lung associated SAgs Kα 1T and ColV. Anti-H2Kb was administered endobronchially in C57BL/6 mice (n=6) and Batf−/− mice (n=6) on days 1, 2, 3, and 6, and weekly thereafter. Sera were collected from the mice on days 15 and 30. ELISA was performed for Abs to SAgs in the serum of mice treated with anti-MHC Abs using purified Kα 1T or ColV as Ag. Loss of Batf results in the decreased concentrations of Abs against Kα 1T (A) and ColV (B) on days 15 and 30 compared with WT mice treated with anti-H2Kb Ab. The data are shown as mean ± SEM fold change observed. P-values were calculated with the Student's t-test; error bars represent SEM. Mean results from two independent experiments are shown (n=3/group).

Batf deficiency leads to decreased production of pro-inflammatory cytokines and chemokines

Inflammatory cytokines and chemokines have been demonstrated to be important in promoting cellular infiltration into the lungs leading to epithelial hyperplasia following MHC I treatment (4). In order to determine the role of Batf in the induction of cytokines and chemokines following administration of anti-MHC, we analyzed the expression of IL-6, IL-23, IL-17, IL-1β, FGF-6, and CXCL-12 in the lungs by qRT- PCR. Analysis of the lungs on day 15 demonstrated that there was a significant reduction in the expression of IL-6 (0.6±0.2 vs. 4.6±0.5, p<0.01), IL-23 (0.6±0.1 vs. 3.7±0.4, p<0.01), IL-17 (0.3±0.1 vs. 6.0±1.1, p<0.01), IL-1β (0.7±0.1 vs. 5.4±0.8, p<0.01), FGF-6 (1.1±0.1 vs. 4.1±0.5, p<0.01), and CXCL-12 (1.0±0.1 vs. 3.7±1.0, p<0.01) in the lungs of Batf−/− mice when compared to WT (Figure 4). These results strongly suggest an important role for Batf in the activation of inflammatory cytokines and chemokines, which are necessary for the induction of immune responses to SAgs following administration of anti-MHC.

Figure 4.

Figure 4

Open in a new tab

Batf deficiency leads to decreased production of pro-inflammatory cytokines and chemokines. Batf−/− (n=3) or C57BL/6 mice (n=3) were treated with anti-MHC class I Abs day 1, 2 and 3. The expression levels of IL-6, IL-23, IL-17, IL-1β, FGF-6, and CXCL-12 in the lungs were analyzed by qRT-PCR on day 15. The data are shown as mean ± SEM fold change observed, with three mice per group. P-values were calculated with the Student's t-test; error bars represent SEM. Mean results from two independent experiments are shown (n=3/ group)

JAK-STAT signaling is impaired in Batf−/− mice after administration of Abs to MHC

Batf−/− mice display a severe deficiency in Th17 cells (7), and as presented above did not develop OAD following administration of anti-MHC. STAT3 activation by IL-6 has been shown to result in the activation of JAK leading to induction of Th17 response (15, 16). We therefore examined the role of Batf in regulating JAK-STAT signaling cascade. Analysis of JAK2 expression in the lungs of anti-MHC administered Batf−/− mice showed significant reduction (~5 fold) in the expression of JAK2 when compared to WT (0.9±0.3 vs. 4.3±1.1, p<0.01, Figure 5A). Further, analysis of STAT3 and RORγ T expression also demonstrated significant reduction (~5 fold) in the levels of STAT3 (0.8±0.2 vs. 4.2±0.8, p<0.01, Figure 5B) and a ~4.2 fold reduction (0.9±0.2 vs. 4.0±0.6, p<0.01, Figure 5C) in the levels of RORγ T when compared to WT. These results demonstrate that loss of JAK-STAT signaling activation in Batf−/− mice correlate strongly with abrogation of anti-MHC induced OAD.

Figure 5.

Figure 5

Open in a new tab

JAK-STAT signaling is impaired in Batf−/− mice after administration of Abs to MHC class I. The expression levels of JAK2 (A), STAT3 (B), and RORγT (C) in the lungs of Batf−/− mice (n=3) were analyzed by qRT-PCR on day 15 compared to WT (n=3) when administration of anti-MHC. The data are shown as mean ± SEM fold change observed, with three mice per group. P-values were calculated with the Student's t-test; error bars represent SEM. Mean results from two independent experiments are shown (n = 3/ group)

Adoptive transfer of T, B lymphocytes reconstitute the OAD lesions in Batf−/− mice

To confirm the role of T, B lymphocytes in modulating OAD lesions, we performed adoptive transfer with T and B cells, into Batf−/− mice. T cells and B cells were positively selected using MACS beads (Miltenyi Biotec) from splenocytes of C57BL/6 animals. As shown in Figures 6, H&E and Trichrome analysis of the lungs recovered from both T and B lymphocytes adoptively transferred Batf−/− mice on day 30 demonstrated significant OAD lesions. These results support that both T and B cells exert a strong modulating effect toward development of anti-MHC induced OAD lesions.

Figure 6.

Figure 6

Open in a new tab

Representative histology on day 30 from Batf−/− mice that underwent injection i.v. of T and B lymphocytes into mouse tail veins on day-1 along with the first dose of intrabronchial Abs. Anti-H2Kb (200 μg/dose) was administered into the lung on day 1, 2, 3, 6 and then weekly thereafter. Mice were sacrificed on Day 30. H&E and Trichrome staining of the lungs. x100. Experiments were performed with three mice per group.

MiR-301a was up-regulated and correlated with down-regulation of PIAS3 expression following administration of Abs to MHC

It has been reported that miR-301a contribute to the development of Th17 subset via targeting PIAS3, a potent inhibitor of the IL-6/23-STAT3 pathway (17). Therefore, we quantitated the expression of miR-301a in WT and Batf−/− mice following administration of anti-MHC. After administration of anti-H2Kb, miR-301a in Batf−/− mice was increased on day 2, and maintained the level till day 30 compared to isotype. In contrast, miR-301a was induced significantly on day 1 in the WT animals following anti-MHC administration which further increased till day 7 compared to animals treated with Isotype. However, miR-301a in Batf−/− mice was significantly lower on days 7 and 30 compared to WT following anti-MHC administration (Figure 7A). Further qRT-PCR confirmed that mRNA expression of the PIAS3 was also down-regulated in anti-H2Kb treated WT mice at day 7 and 15 compared to treatment with isotype Abs (Figure 7B). In contrast, there was no significant change in PIAS3 mRNA levels in Batf−/− mice after administration of anti-MHC on days 7 and 15 (Figure 7B). These results demonstrate that up-regulation of miR-301a leading to down-regulation of PIAS3 expression is important for Th17 response leading to immune response to SAgs resulting in OAD.

Figure 7.

Figure 7

Open in a new tab

MiR-301a was up regulated and correlated with down regulation of PIAS3 expression following administration of Abs to MHC class I. (A). RT-PCR analysis of the expression of miR-301a in lung tissues from Batf−/− (n=3) and WT mice (n=3) after 1, 2, 3, 7, 15, 30 days after anti-H2Kb or isotype Abs administration. (B). qRT-PCR was performed to test the mRNA level of PIAS3 expression in Batf−/− and WT mice treated with anti-MHC compared to mice receiving isotype Abs. The data are shown as mean ± SEM fold change observed, with three mice per group. P-values were calculated with the Student's t-test; Mean results from two independent experiments are shown (n = 3/ group).

MiR-301a was higher abundance in LTxR with de novo development of DSA

To determine whether miR-301a is increased following DSA development in human LTxR, we obtained BAL cells from 15 LTxR with DSA and 10 stable LTxR with no detectable DSA at the time of routine bronchoscopy. Isolated BAL cells were analyzed for the expression of miR301a by TaqMan miRNA assay and its target PIAS3 by qRT-PCR. There was a significant higher level of miR-301a expression in LTxR with DSA compared to LTxR without DSA (p=0.022, Figure 8A). In addition, a significant concomitant lower level in PIAS3 mRNA expression was noted in LTxR who developed DSA when compared to stable LTx patients without DSA (p=0.011, Figure 8B). Therefore, miR-301a and PIAS3 mRNA expression in LTxR with de novo development of DSA to HLA corresponds well with the results obtained in the murine model of OAD induced by anti-MHC. These strongly suggest an important role for the miR-301a in regulating the immune response leading to chronic rejection following human LTx.

Figure 8.

Figure 8

Open in a new tab

miRNA301a was up regulated in LTx recipients with de novo development of DSA. (A). Validation of miR-301a in LTx recipients with de novo development of DSA (n=15) compared to stable LTx recipients (n=10). Expression fold changes of miR-310a were calculated by 2−ΔΔCt method, normalized to the expression of U6. Expression fold changes of miR-310a in DSA LTxR were compared to those of stable LTxR. (B). RT-PCR of PIAS3 mRNA levels in LTxR with de novo development of DSA (n = 15) compared to LTxR without DSA (n=10). P-values were calculated with the Mann-Whitney test; error bars represent SEM.

Discussion

Induction of OAD by anti-MHC recreates a relevant pre-clinical model that in human LTx development of Abs to donor mismatched HLA, which often precedes development of BOS (4). In this study, we examined the potential role of Batf in the induction of immune response to lung associated SAgs and development of anti-MHC induced OAD. We demonstrated that Batf deficiency resulted in significant reduction in cellular infiltration, epithelial metaplasia, and fibrosis following administration of anti-MHC (Figure 1). BATF and the closely related BATF3 of the ATF family contain only a basic region and leucine zipper, and act as inhibitors of activator protein-1 activity (18). BATF by regulating the expression of RORγt and its targets leads to differentiation of Th17 cells (7). The important role for IL-17 has also been demonstrated in the development of chronic rejection following human LTx (19, 20). Studies from Wilkes et al have shown that ColV-specific CD4+ T cells secreting IL-17 are present in peripheral blood of LTxR with BOS (19). Studies from our laboratory demonstrated that BOS patients had higher frequency of IL-17-producing Kα1T reactive CD4+ T cells (14). Results presented in this study for the first time demonstrated that Batf deficiency leads to decreased production of IL-6, IL-17, and IL-23 (Figure 4) following administration of anti-MHC with marked reduction in OAD lesions (Figure 1). In addition, Batf deficiency also resulted in marked decrease of IL6-JAK-STAT3 signaling activity (Figure 5). These data demonstrate that there is a strong relationship between BATF and STAT3-mediated development of Th17 cells following IL-6/IL-23 expression triggered by Abs to MHC. Taken together our results strongly support the conclusion that Batf deficiency leads to abrogation of OAD through blocking of Th17 response specific to lung associated SAgs.

BATF proteins (BATF, BATF2, and BATF3) can compete with Fos for partnering with Jun and generate bZIP dimers that inhibit the transcription of AP1 reporter genes (21). The transcription factor BATF controls the Th17 differentiation by regulating expression of the transcription factor RORγt itself and RORγt target genes such as IL-17 (7). BATF represses the effector function of exhausted CD8+T cells (22) and regulates metabolism and/or epigenetics in CD8+T cells via the histone deacetylase Sirt1 (23). BATF was also found to orchestrate the developmental transition from a naive state to an effector state in CD8+T cells (24). Loss of Batf leads to the defects in naive CD8+T cells differentiation into effector CD8+T cells, which could partially explain why Batf−/− mice are protected from anti-MHC induced OAD. BATF directly controlled expression of the transcription factors Bcl-6 and c-Maf, both of which are needed for development of Tfh cells (10). In B cells, Batf expression was induced by both IL-6 signaling via STAT3 or stimulation with lipopolysaccharide (11, 25). We postulate that Batf−/− mice lack Tfh cells and have intrinsic B cell defects potentially limiting OAD severity. The results from our study for the first time demonstrated that lack of Batf results in loss of induction of SAg reactive T cells. IL-10 producing T cells specific for Kα1T and ColV (Treg cells) were decreased in WT after MHC I Ab treatment. IL-4 and IFNγ-producing cells were increased after MHC I Ab treatment, which demonstrated that Th1 and Th2 cells were increased after administration of anti-HLA. In addition, our results also demonstrated that Batf deficiency leads to decreased humoral immune responses against lung associated SAgs (Figure 3). Batf deficient mice did not produce IgG1 or IgG3, except IgM in response to immunization with a T-dependent antigen, suggesting a global defect in ability to produce antigen specific IgG (11). In Figure 3, Ab to Kα1T and ColV was produced in Batf−/− mice after challenging with anti-MHC I Ab, although delayed. We further measured IgG and IgM subtypes for auto-Abs against Kα1T and ColV in C57BL/6 and Batf−/− mice treated with anti-MHC on day 30. Batf deficient mice did not produce IgG anti-Kα1T and anti-ColV in response to immunization with anti-MHC, however, there was IgM Abs to Kα1T and ColV (Figure S1). Analysis of the cytokine expression profile in the lungs following anti-MHC administration also demonstrated significant reduction in the expression of IL-6, IL-23, IL-17, IL-1β, FGF-6, and CXCL-12 in Batf−/− mice (Figure 4). IL-6 has been shown to induce the development and expansion of Th17 cells that are involved in the immune responses against SAgs resulting in many autoimmune diseases (26). FGF-6 was induced by overexpression of miR-144, a critical contributing factor in the development of BOS after human LTx (27). CXCL12 is a ligand of the chemokine receptor, CXCR4, and plays an important role in pulmonary fibrosis (28). Therefore, results presented in this manuscript clearly demonstrated that BATF is essential for immune responses leading to activation of both humoral and cellular immunity against lung associated SAgs leading to development of anti-MHC induced OAD.

Recent studies have demonstrated that miRNA can regulate several immune functions leading to natural killer T-cell development (29), Th cells differentiation (17, 30) and autoimmunity (31, 32). One of the miRNAs, miR-301a has been shown to regulate gene expressions for RORα, RORγt, and Aryl hydrocarbon receptor required for IL17 production (17). It has been demonstrated that down-regulation of miR-301a can lead to marked loss of STAT3 phosphorylation resulting in blocking of signals for both IL6 and IL23 (17). The activation of IL-6/23 induced STAT3 pathway is a critical and specific step required for generation and maintenance of Th17 cells (33). One of the molecular mechanisms by which miR-301a regulates the Th17 cell development is through interference with PIAS3 (17). PIAS3 is an E3 SUMO ligase that has been shown to act as both a transcriptional coactivator and a corepressor for many transcription factors (34). The PIAS3 activity has been linked primarily to the inhibition of STAT3 (35). PIAS3 along with another PIAS member (PIAS1) has been recently demonstrated to be a critical factor in Th17 and T regulatory cell development and function, and have strong implications in the pathogenesis of EAE (17, 36). So far little is known about the role of PIASs in induction of immune response to lung associated SAgs and immunopathogenesis of OAD following administration of anti-MHC. We present evidence for the first time that in animals with anti-MHC induced OAD as well as human LTxR with de novo development of DSA there is marked increase in miR-310a expression which is accompanied by concomitant down-regulation of PIAS3 (Figure 7 and 8). In contrast, Batf−/− animals following administration of anti-MHC as well as LTxR with undetectable DSA did not demonstrate significant reduction of both miR-301a and enhancement of PIAS3 mRNA levels, which strongly support our conclusion that decrease of miR-301a up-regulates PIAS3 leading to reduction in Th17 activation and IL17 secretion, thus abrogating OAD development. Since we have not performed specific ablation of miR-301a in vivo, the role of miR-301a in preventing OAD development can't be verified at the present time.

In conclusion, we, for the first time, demonstrated that BATF is indispensable for induction of T-cell response through either antigen presentation or secretion of cytokines that can lead to the induction of immune response to lung associated SAgs and development of OAD following administration of anti-MHC. Our findings both in the animal model of OAD induced by anti-MHC and human LTxR with de novo development of DSA also indicate an important role of miR-301a and its target, PIAS3, in Th17 response leading to chronic rejection. We propose that targeting BATF will inhibit the development of immune response to lung associated SAgs and therefore is a potential target for preventing development of chronic rejection following human LTx.

Supplementary Material

Figure S1

IgG and IgM subtypes in auto-Abs to Kα1T and ColV. ELISA was performed to measure the IgG and IgM in sera of Batf−/− and WT mice treated with anti-MHC and Isotype for 30 days. Shown are mean results from three mice per group (n = 3) assayed in duplicate. Error bars indicate SEM.

Table S1
Table S2

Acknowledgements

We thank Billie Glasscock for her assistance in preparing this manuscript. This work is supported by NIH HL092514 and HL056643.and the BJC foundation (TM).

Abbreviations

Abs

antibodies

ATF

activating transcription factor-like

BAL

Bronchoalveolar lavage

BATF

B cell, activating transcription factor-like

BOS

Bronchiolitis Obliterans Syndrome

ColV

Collagen V

DSA

donor specific antibodies

EAE

experimental autoimmune encephalomyelitis

H&E

hematoxylin and eosin

JAK2

Janus kinase 2

Kα1T

K-α1 tubulin

LTx

lung transplantation

LTxR

lung transplant recipient

miRNA

microRNA

OAD

Obliterative Airway Disease

qRT-PCR

quantitative Real Time PCR

RORα

Retinoid-related orphan receptor alpha

RORγT

Retinoid-related orphan receptor gamma-T

SAg

self-antigen

Tfh cells

T follicular helper cells

Th

T helper

Footnotes

Disclosures The authors of this manuscript have no financial conflict of interest to disclose as described by the American Journal of Transplantation.

Description of Supporting Information Additional Supporting Information may be found in the online version of this article.

References

  • 1.Sundaresan S, Trulock EP, Mohanakumar T, Cooper JD, Patterson GA. Prevalence and outcome of bronchiolitis obliterans syndrome after lung transplantation. Washington University Lung Transplant Group. Ann Thorac Surg. 1995;60(5):1341–1346. doi: 10.1016/0003-4975(95)00751-6. discussion 1346–1347. [DOI] [PubMed] [Google Scholar]
  • 2.Palmer SM, Davis RD, Hadjiliadis D, Hertz MI, Howell DN, Ward FE, et al. Development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome. Transplantation. 2002;74(6):799–804. doi: 10.1097/00007890-200209270-00011. [DOI] [PubMed] [Google Scholar]
  • 3.Jaramillo A, Smith MA, Phelan D, Sundaresan S, Trulock E, Lynch J, et al. Temporal relationship between the development of anti-HLA antibodies and the development of bronchiolitis obliterans syndrome after lung transplantation. Transplant Proc. 1999;31(1–2):185–186. doi: 10.1016/s0041-1345(98)01495-x. [DOI] [PubMed] [Google Scholar]
  • 4.Fukami N, Ramachandran S, Saini D, Walter M, Chapman W, Patterson GA, et al. Antibodies to MHC class I induce autoimmunity: role in the pathogenesis of chronic rejection. J Immunol. 2009;182(1):309–318. doi: 10.4049/jimmunol.182.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Aronheim A, Zandi E, Hennemann H, Elledge SJ, Karin M. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol. 1997;17(6):3094–3102. doi: 10.1128/mcb.17.6.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Dorsey MJ, Tae HJ, Sollenberger KG, Mascarenhas NT, Johansen LM, Taparowsky EJ. B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family. Oncogene. 1995;11(11):2255–2265. [PubMed] [Google Scholar]
  • 7.Schraml BU, Hildner K, Ise W, Lee WL, Smith WA, Solomon B. The AP-1 transcription factor Batf controls T(H)17 differentiation. Nature. 2009;460(7253):405–409. doi: 10.1038/nature08114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Martinez GJ, Dong C. BATF: bringing (in) another Th17-regulating factor. J Mol Cell Biol. 2009;1(2):66–68. doi: 10.1093/jmcb/mjp016. [DOI] [PubMed] [Google Scholar]
  • 9.Martinez GJ, Nurieva RI, Yang XO, Dong C. Regulation and function of proinflammatory TH17 cells. Ann N Y Acad Sci. 2008;1143:188–211. doi: 10.1196/annals.1443.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ise W, Kohyama M, Schraml BU, Zhang T, Schwer B, Basu U, et al. The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Nat Immunol. 2011;12(6):536–543. doi: 10.1038/ni.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Betz BC, Jordan-Williams KL, Wang C, Kang SG, Liao J, Logan MR, et al. Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J Exp Med. 2010;207(5):933–942. doi: 10.1084/jem.20091548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tiriveedhi V, Takenaka M, Sarma NJ, Gelman AG, Mohanakumar T. Anti-major histocompatibility complex-induced obliterative airway disease: selective role for CD4 and CD8 T cells in inducing immune responses to self-antigens. J Heart Lung Transplant. 2013;32(7):714–722. doi: 10.1016/j.healun.2013.04.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Bharat A, Narayanan K, Street T, Fields RC, Steward N, Aloush A, et al. Early posttransplant inflammation promotes the development of alloimmunity and chronic human lung allograft rejection. Transplantation. 2007;83(2):150–158. doi: 10.1097/01.tp.0000250579.08042.b6. [DOI] [PubMed] [Google Scholar]
  • 14.Hachem RR, Tiriveedhi V, Patterson GA, Aloush A, Trulock EP, Mohanakumar T. Antibodies to K-alpha 1 tubulin and collagen V are associated with chronic rejection after lung transplantation. Am J Transplant. 2012;12(8):2164–2171. doi: 10.1111/j.1600-6143.2012.04079.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Senga T, Iwamoto T, Humphrey SE, Yokota T, Taparowsky EJ, Hamaguchi M. Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells. Oncogene. 2002;21(53):8186–8191. doi: 10.1038/sj.onc.1205918. [DOI] [PubMed] [Google Scholar]
  • 16.Harris TJ, Grosso JF, Yen HR, Xin H, Kortylewski M, Albesiano E, et al. Cutting edge: An in vivo requirement for STAT3 signaling in TH17 development and TH17-dependent autoimmunity. J Immunol. 2007;179(7):4313–4317. doi: 10.4049/jimmunol.179.7.4313. [DOI] [PubMed] [Google Scholar]
  • 17.Mycko MP, Cichalewska M, Machlanska A, Cwiklinska H, Mariasiewicz M, Selmaj KW. MicroRNA-301a regulation of a T-helper 17 immune response controls autoimmune demyelination. Proc Natl Acad Sci U S A. 2012;109(20):E1248–1257. doi: 10.1073/pnas.1114325109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Williams KL, Nanda I, Lyons GE, Kuo CT, Schmid M, Leiden JM, et al. Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus. Eur J Immunol. 2001;31(5):1620–1627. doi: 10.1002/1521-4141(200105)31:5<1620::aid-immu1620>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  • 19.Burlingham WJ, Love RB, Jankowska-Gan E, Haynes LD, Xu Q, Bobadilla JL, et al. IL-17-dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants. J Clin Invest. 2007;117(11):3498–3506. doi: 10.1172/JCI28031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Fan L, Benson HL, Vittal R, Mickler EA, Presson R, Fisher AJ, et al. Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation. Am J Transplant. 2011;11(5):911–922. doi: 10.1111/j.1600-6143.2011.03482.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Echlin DR, Tae HJ, Mitin N, Taparowsky EJ. B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos. Oncogene. 2000;19(14):1752–1763. doi: 10.1038/sj.onc.1203491. [DOI] [PubMed] [Google Scholar]
  • 22.Quigley M, Pereyra F, Nilsson B, Porichis F, Fonseca C, Eichbaum Q, et al. Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF. Nat Med. 2010;16(10):1147–1151. doi: 10.1038/nm.2232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kuroda S, Yamazaki M, Abe M, Sakimura K, Takayanagi H, Iwai Y. Basic leucine zipper transcription factor, ATF-like (BATF) regulates epigenetically and energetically effector CD8 T-cell differentiation via Sirt1 expression. Proc Natl Acad Sci U S A. 2011;108(36):14885–14889. doi: 10.1073/pnas.1105133108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kurachi M, Barnitz RA, Yosef N, Odorizzi PM, DiIorio MA, Lemieux ME, et al. The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells. Nat Immunol. 2014;15(4):373–383. doi: 10.1038/ni.2834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ellyard JI, Vinuesa CG. A BATF-ling connection between B cells and follicular helper T cells. Nat Immunol. 2011;12(6):519–520. doi: 10.1038/ni.2042. [DOI] [PubMed] [Google Scholar]
  • 26.Kimura A, Kishimoto T. IL-6: regulator of Treg/Th17 balance. Eur J Immunol. 2010;40(7):1830–1835. doi: 10.1002/eji.201040391. [DOI] [PubMed] [Google Scholar]
  • 27.Xu Z, Ramachandran S, Gunasekaran M, Zhou F, Trulock E, Kreisel D, et al. MicroRNA-144 dysregulates the transforming growth factor-beta signaling cascade and contributes to the development of bronchiolitis obliterans syndrome after human lung transplantation. J Heart Lung Transplant. 2015;34(9):1154–1162. doi: 10.1016/j.healun.2015.03.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lama VN, Phan SH. The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond. Proc Am Thorac Soc. 2006;3(4):373–376. doi: 10.1513/pats.200512-133TK. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Fedeli M, Napolitano A, Wong MP, Marcais A, de Lalla C, Colucci F, et al. Dicer-dependent microRNA pathway controls invariant NKT cell development. J Immunol. 2009;183(4):2506–2512. doi: 10.4049/jimmunol.0901361. [DOI] [PubMed] [Google Scholar]
  • 30.Wang H, Flach H, Onizawa M, Wei L, McManus MT, Weiss A. Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210. Nat Immunol. 2014;15(4):393–401. doi: 10.1038/ni.2846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Cobb BS, Nesterova TB, Thompson E, Hertweck A, O'Connor E, Godwin J, et al. T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer. J Exp Med. 2005;201(9):1367–1373. doi: 10.1084/jem.20050572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Muljo SA, Ansel KM, Kanellopoulou C, Livingston DM, Rao A, Rajewsky K. Aberrant T cell differentiation in the absence of Dicer. J Exp Med. 2005;202(2):261–269. doi: 10.1084/jem.20050678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem. 2007;282(13):9358–9363. doi: 10.1074/jbc.C600321200. [DOI] [PubMed] [Google Scholar]
  • 34.Yagil Z, Nechushtan H, Kay G, Yang CM, Kemeny DM, Razin E. The enigma of the role of protein inhibitor of activated STAT3 (PIAS3) in the immune response. Trends Immunol. 2010;31(5):199–204. doi: 10.1016/j.it.2010.01.005. [DOI] [PubMed] [Google Scholar]
  • 35.Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, et al. Specific inhibition of Stat3 signal transduction by PIAS3. Science. 1997;278(5344):1803–1805. doi: 10.1126/science.278.5344.1803. [DOI] [PubMed] [Google Scholar]
  • 36.Liu B, Tahk S, Yee KM, Fan G, Shuai K. The ligase PIAS1 restricts natural regulatory T cell differentiation by epigenetic repression. Science. 2010;330(6003):521–525. doi: 10.1126/science.1193787. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

IgG and IgM subtypes in auto-Abs to Kα1T and ColV. ELISA was performed to measure the IgG and IgM in sera of Batf−/− and WT mice treated with anti-MHC and Isotype for 30 days. Shown are mean results from three mice per group (n = 3) assayed in duplicate. Error bars indicate SEM.

NIHMS758191-supplement-Figure_S1.tif (185.4KB, tif)
Table S1
NIHMS758191-supplement-Table_S1.docx (34.7KB, docx)
Table S2
NIHMS758191-supplement-Table_S2.docx (32.8KB, docx)

RESOURCES