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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: J Immunol. 2022 Sep 16;209(9):1788–1795. doi: 10.4049/jimmunol.2200245

Cross-Regulation of FBox Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection

Antu Das #,1, Xingan Wang #,1, Jianxin Wei #, Aki Hoji #, Tiffany A Coon #, Iulia Popescu #, Mark Brown #, Sheila Frizzell #, Carlo J Iasella †, Kentaro Noda *, John Sembrat #, Kaitlyn Devonshire #, Stefanie J Hannan #, Mark E Snyder #, Joseph Pilewski #, Pablo G Sanchez *, Divay Chandra #, Rama K Mallampalli §, Jonathan K Alder #, Bill B Chen #,, John F McDyer #
PMCID: PMC9588753  NIHMSID: NIHMS1831441  PMID: 36113884

Abstract

Chronic lung allograft dysfunction(CLAD) is the major barrier to long-term survival in lung transplant recipients(LTRs). Evidence supports Type-1 alloimmunity as the predominant response in acute/chronic lung rejection, the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F box E3-ligase system, FBXO3(pro-inflammatory) and FBXL2(anti-inflammatory; regulates tumor necrosis factor receptor-associated factor(TRAF) protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c>C57BL/6(acute rejection; day-10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ and TRAF proteins 1–5 compared to isografts. In the acute model, treatment with costimulation-blockade (MR1/CTLA4-Ig)resulted in attenuated FBXO3, preserved FBXL2 and substantially reduced T-bet, IFN-γ and TRAFs 1–5, consistent with a key role for Type-1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3:FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared to isografts or costimulation-blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1>DBA/2J(day-28) we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein, and similar findings from LTR lungs with CLAD versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and co-immunoprecipitated with T-bet on pull-down experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in MLE cells. In testing Type-1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3-ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

INTRODUCTION

Lung transplantation remains the only therapeutic option for select patients with end-stage lung disease. Despite advances in immunosuppression and surgical techniques, long-term survival in lung transplant recipients (LTRs) lags behind other solid organ recipients, with a median survival of only 5.7 years(1, 2). Chronic lung allograft dysfunction (CLAD) is the major long-term barrier to survival, with bronchiolitis obliterans syndrome (BOS) the predominant clinical phenotype and acute cellular rejection (ACR) the most significant risk-factor for BOS/CLAD in LTRs(3). We and others have shown that Type-1 immunity is the predominant pathway for allograft rejection in mouse models of lung transplant, including the mouse orthotopic left lung transplant model(4, 5). More recently, we have shown that CLAD in LTRs is marked by an airway transcriptome with a Type-1 immune signature and intragraft donor-specific alloimmune T cell responses with interferon (IFN)-γ, tumor necrosis factor (TNF)-α and CD107a as predominant(6). However, the regulatory mechanisms that govern Type-1 immune responses, and in particular the key Type-1 transcriptional factor, T-bet(7), during lung transplant rejection have not been fully elucidated.

In prior studies, we have demonstrated a novel combinatorial pro-inflammatory E3 ligase subunit, F-Box Protein 3 (FBXO3), that degrades an anti-inflammatory E3 ligase subunit, F-Box And Leucine Rich Repeat Protein 2 (FBXL2)(8). Previous studies from our group have shown that FBXL2, a member of the SCF (Skip-Cullin1-F-box protein) E3 ligase family, is a critical checkpoint for cell cycle progression factors and the TNF receptor-associated factor adaptor proteins (TRAFs), which are upstream to numerous inflammatory cytokines including the Type-1 cytokines, IFN-γ and TNF-α(8–11). Based on this, we hypothesized that the FBXO3:FBXL2 balance was an important regulator in lung allograft rejection, as we have shown this pathway to be important in other pulmonary processes such as acute lung injury and influenza viral pneumonia(8, 12). Here, using the mouse orthotopic lung transplant (OLT) model, we show that the pro-inflammatory FBXO3 protein is upregulated during acute and chronic lung allograft rejection, resulting in decreased levels of the anti-inflammatory FBXL2 protein, and that this pathway is alloimmune inflammation-dependent, as it is blocked using T cell co-stimulation-blockade. We further show that FBXL2 is an immune regulator of T-bet through polyubiquitination and that the Type-1 cytokine, TNF-α, cross-regulates FBXL2.

MATERIALS AND METHODS

Mice

C57BL/6J, B6DBAF1/J, DBA/2J and BALB/c mice were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were housed in the University of Pittsburgh animal facilities under specific pathogen-free conditions. Animals were between 7 and 9 weeks of age and around 25 g in weight at the time of experiments. The University of Pittsburgh Institutional Animal Care and Use Committee approved all procedures.

Mouse Orthotopic Lung Transplant

Allogeneic or isogenic transplantations were performed in the BALB/c → C57BL/6J, C57BL/6J → C57BL/6J, B6DBAF1/2J→ DBA/2J or DBA/2J→ DBA/2J strain combinations. Donor mice were sedated with etomidate (1 mg, intraperitoneally), intubated, and maintained on inhaled isoflurane until they were sacrificed. Recipients were both initially sedated and maintained on inhaled isoflurane. OLT was performed using a cuffed technique as previously described(13–15). Mice received subcutaneous buprenorphine (0.03–0.05 mg/kg) before extubation and every 6 hours thereafter as needed. Mice were treated with or without costimulatory blockade CD154/CD40 (MR1) (250 µg at day 0) and cytotoxic T lymphocyte antigen 4-Ig (CTLA4Ig) (250 µg at day 2). Animals were euthanized for analysis at 10 or 28 days after transplant.

Mouse T cell Isolation

The EasySep™ Mouse T Cell Isolation Kit is used to isolate T cells from single-cell suspensions of allograft lungs by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies directed against non-T cells and streptavidin-coated magnetic particles. Labeled cells are separated using an EasySep™ magnet without the use of columns.

Cell lines

The mouse lung epithelial (MLE12) cell line and Jurkat T cell line were purchased from ATCC (Manassas, VA). MLE12 cells were cultured in a CO2 incubator (5% CO2–95% air) at 37 °C in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F-12; Invitrogen), supplemented with 2% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 1% l-glutamine, 1% HEPES, 1% insulin/transferrin/sodium selenite, 0.01% β-estradiol, and 0.01% hydrocortisone. Jurkat cells were cultured in RPMI medium supplemented with 10% fetal bovine serum. MLE12 cells were treated with or without varying doses of the Type-1 cytokines TNF-α (0, 10 and 100 ng/ml), IL-1β (0, 1, 10 and 100 ng/ml) and IFN-γ (0, 10 and 100 U/ml) at 0h, 4h, 8h and 24h.

Human lung samples

The University of Pittsburgh Institutional Review Board approved this study. After obtaining informed consent from the subjects, we collected explant lung tissue obtained from subjects diagnosed with CLAD/BOS and undergoing re-transplantation (clinically diagnosed and confirmed) or healthy donor lung declined for transplant. Lungs were homogenized and proteins assayed for FBXO3, FBXL2, T-bet, and IFN-γ immunoblots.

Immunoprecipitation and immunoblotting

Cell lysates in 150 μl of lysis buffer (20 mm Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM β-glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 μg/ml of protease inhibitors, 1 μg/ml of aprotinin, 1 μg/ml of leupeptin, and 1 μg/ml of pepstatin) were sonicated on ice for 12 s and centrifuged at 10,000 × g for 10 min at 4°C in a microcentrifuge. For immunoprecipitation, equal amounts of cell lysates (1 mg) were incubated with 5 μg/ml of specific primary antibodies overnight at 4°C followed by the addition of 40 μl of protein A/G-agarose for 4h at 4°C. For immunoblotting, equal amounts of supernatant (20 μg) were subjected to 10% SDS-PAGE gels, transferred to nitrocellulose membranes, blocked with 5% (w/v) nonfat milk in TBST (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween 20) for 1h, and incubated with primary antibodies in 5% (w/v) BSA in TBST for 1–2h. The membranes were washed at least three times with TBST at 10-min intervals followed by a 1-h incubation with mouse, rabbit, or goat horseradish peroxidase-conjugated secondary antibody (1:500). The membranes were developed with an enhanced chemiluminescence detection system according to manufacturer’s instructions(16, 17).

RT-PCR

RNA was isolated from cells using RNeasy Plus Mini Kits (Qiagen, Hilden, Germany) per the protocol provided. Isolated RNAs were immediately converted to cDNA using High-Capacity RNA-to-cDNA Kits (Life Technologies, Carlsbad, CA) after their concentrations were measured.

The cDNA was then amplified using primers purchased from ThermoFisher (Mm00618190_m1) using TaqMan fast advance master mix. Quantification of gene expression was performed with a sequence-detection system (CFX96 Real-Time PCR Detection System; Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s protocol. Target gene mRNA expression was calculated and was normalized to that of the housekeeping gene 18S rRNA. The experiment was duplicated, and the mean and standard error were calculated.

In vitro ubiquitination assay

The ubiquitination of substrates were performed in a volume of 25 μl containing 50 mM Tris pH 7.6, 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 1.5 ng/μl E1 (Promega, Madison, WI), 10 ng/μl Ubc5 (Promega, Madison, WI), 10 ng/μl Ubc7 (Promega, Madison, WI), 1 μg/μl ubiquitin (Promega, Madison, WI), 1 μM ubiquitin aldehyde (Promega), 4–16 μl of purified Cullin1 (R&D system, Minneapolis, MN), Skp1 (R&D system), Rbx1 (R&D system) and in vitro–synthesized Fbxl2 or T-bet. Reaction products were processed for Flag (MilliporeSigma, Burlington, MA) and HA (Cell Signaling technology, Danvers, MA) immunoblotting.

Histology and Immunohistochemistry

The lung tissues were removed and fixed in 10% formalin and embedded in paraffin. Tissue sections of 5 μm thickness were stained with hematoxylin and eosin (H&E) to determine lung architecture. Immunohistochemistry (IHC) staining was performed according to standard laboratory procedures using the following primary antibodies: rabbit anti-FBXL2 polyclonal antibody (1:100; Aviva Systems Biology, San Diego, CA), rabbit anti-FBXO3 polyclonal antibody (1:100; Santa Cruz Biotechnology Inc., Dallas, Tx) and rabbit anti-CD3 monoclonal antibody (1:150; Abcam, Boston, MA). Images were taken using upright microscope (Nikon Eclipse Ni-E).

Statistics

Immunoblot analyses were quantified using ImageJ processing software. Data were analyzed using Graphpad and are presented as mean ± standard deviation. The student’s 2-tailed t test was used to determine P values when comparing 2 groups. A P-value <0.05 was considered significant.

RESULTS

The change in balance of FBXO3:FBXL2 during mouse acute lung allograft rejection is Type-1 alloimmunity-dependent

Using the mouse left OLT model, we assessed FBXO3, FBXL2, type I immune components, IFN-γ, T-bet and the TRAF1–5 proteins in acute rejection at day 10 posttransplant. We performed western blot analysis and found significant increase in the balance of FBXO3:FBXL2 protein expression during acute allograft lung rejection compared to isograft control lungs (Figure 1 A, B). We also observed striking upregulation of T-bet and IFN-γ protein expression in lung allografts compared to isografts, consistent with other studies. Further we observed significant protein expression of the TRAFT1–5 proteins during allograft rejection compared to isografts. Treatment with the costimulatory blockade targeting CD154/CD40 interactions (MR1) and cytotoxic T lymphocyte antigen 4-Ig (CTLA4Ig), resulted in a marked reduction of FBXO3:FBXL2 protein balance similar to levels seen in isografts. Similarly, T-bet and IFN-γ protein expression, as well as the TRAF1–5 proteins, were significantly reduced to baseline levels following co-stimulation blockade, comparable to those observed in isograft lungs. We also assessed FBXL2:FBXO3 balance and T-bet protein expression in total T cells isolated from mouse allograft lungs treated with or without costimulatory blockade. We performed western blot and found an increase in the balance of FBXL2:FBXO3 protein expression in T cells during acute allograft lung rejection along with significant increase in T-bet protein expression. Treatment with costimulatory blockade resulted a reduction of FBXO3:FBXL2 protein balance compared to the allografts. T-bet protein expression also significantly reduced following co-stimulation blockade compared to the allografts (Figure 1C-D). Further we performed the western blot to assess the balance of FBXL2:FBXO3 protein expression following a dose-dependent stimulation with anti-CD3/CD28 for 24 h. We observed markedly increased in FBXO3/T-bet protein expression coupled with decreased FBXL2 protein expression (Figure 1E), suggesting that the FBXO3:FBXL2 balance regulates T-bet protein stability and potentially plays a role in T cell immunity.

Figure 1. FBXO3:FBXL2 balance in mouse acute lung allograft rejection is Type-1 alloimmunity-dependent.

Figure 1.

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(A) The protein expression of FBXO3, FBXL2, T- bet, IFN-γ and TRAFs in isograft and allograft mouse lungs with or without MR1-CTLA4Ig treatment at day 10, as examined by western blot assay. (B) Densitometric quantification of FBXO3, FBXL2, T- bet, IFN-γ and TRAFs proteins detected by western blot for (A), n=3 mice/group. (C) The protein expression of FBXL2, FBXO3 and T- bet in Total T cells isolated from allograft mouse lungs with or without MR1-CTLA4Ig treatment at day 10, as examined by western blot assay. (D) Densitometric quantification of FBXL2, FBXO3 and T- bet proteins detected by western blot for (C), n=3 mice/group. (E) The protein expression of FBXO3, FBXL2 and T- bet in jurkat cells (CD4 T cells) stimulated with αCD3/CD28 in a dose-dependent manner for 24h. Cumulative data showing densitometry of the isograft (light grey columns), allograft (black columns) and allograft with co-stimulation blockade (dark grey columns). All results are representative of three replicate experiments, using Student t test. *p < 0.05, **p < 0.005.

Next, we performed IHC at day 10 to further assess FBXO3:FBXL2 protein balance during acute rejection compared to isografts as shown in Figure 2. Shown are higher staining levels for FBXO3 during rejection and a reduction in FBXL2 levels around the airway. The addition of co-stimulation blockade markedly reduced FBXO3 staining around the airways and resulted in intensified staining of FBXL2 (Figure 2). Together these data indicate that changes in the FBXO3:FBXL2 protein balance as well as the type I immune molecules T-bet/IFN-γ and the TRAF1–5 proteins are upregulated during acute cellular rejection and are T cell co-stimulation blockade dependent, shown previously to abrogate type I alloimmune responses.

Figure 2. FBXO3 and FBXL2 and expressed in airway epithelia and mononuclear cells during mouse acute rejection.

Figure 2.

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(A)(C) Immunohistochemical staining of FBXO3 and FBXL2 in isografts (B6→B6) (left column), allografts (BALB/c→B6) (middle column) and allografts with MR1-CTLA4Ig treatment (right column). (B)(D) Densitometry analysis of Immunohistochemical staining of FBXO3 and FBXL2 for (A) & (C). Data are representative of n= 4 mice/group.

Persistence of elevated FBXO3:FBXL2 protein balance during chronic lung allograft rejection

We next evaluated the FBXO3:FBXL2 ubiquitin pathway in chronic lung allograft rejection. For this we used the previously described mouse OLT strain combination, B6/DBA/2J F1→DBA/2J allografts at 28 days post-transplantation(18). We observed similar elevated levels by western blot and densitometry analyses of FBXO3, IFN-γ and T-bet along with downregulation of FBXL2 expression in allograft lungs compared to isografts (DBA/2J→DBA/2J) or DBA/2J native lungs from allograft recipients (right lung) (Figure 3A-B). Notably, we observed an inverse relationship between higher FBXL2 levels in isografts and native lungs and lower T-bet levels (Figure 3B). The eosin and hematoxylin staining showed a mononuclear cell infiltration surrounding the airway with extended inflammation into the alveolar spaces of allograft lungs in contrast to isograft lungs (Figure 3C). We further assessed IHC in this chronic rejection model and found persistent high-level staining of FBXO3>FBXL2 during chronic allograft rejection in contrast to FBXL2>FBXO3 in isograft lungs (Figure 3D). We also observed CD3+ lymphocytes surrounding the airway epithelium where both FBXL2 and FBXO3 staining was detected (Figure 3F). Last we assessed explants from human lung transplant recipients with end-stage chronic rejection or CLAD, to evaluate the FBXO3/FBXL2 ubiquitin pathway. Similar to our findings in the mouse OLT model, we found FBXO3>FBXL2 levels in explanted CLAD lungs from patients undergoing re-transplantation (Supplemental Table 1), by western blot and densitometry quantification (Figure 3E-F). Additionally, we found elevated protein levels of IFN-γ, and T-bet in CLAD explanted lungs compared to controls. Together these data demonstrate persistently elevated FBXO3:FBXL2 protein balance in mouse and human chronic lung allograft rejection. Moreover, we observed an inverse relationship between FBXL2 and T-bet.

Figure 3. Persistence of elevated FBXO3:FBXL2 protein balance during chronic rejection in mouse and human allografts.

Figure 3.

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(A) Representative lung allografts from untreated mice (B6DBAF1/J>DBA/2J) at day 28 for FBXO3, FBXL2, T- bet/IFN-γ compared to isografts or native lung from allograft mice using lung lysates for western blot analyses from n=3 mice/group; (B) Densitometry analysis for FBXO3, FBXL2, T-bet, and IFN-γ quantification from (A). Cumulative data showing densitometry of the isograft (light grey columns), allograft (black columns) and native lung (dark grey columns). All results are representative of three replicate experiments using Student t test. *p < 0.05, **p < 0.005. (C) Hematoxylin and eosin (H&E)–staining of lung sections from isograft (DBA/2J>DBA/2J) (left) and allograft (B6DBAF1/J>DBA/2J) (right) at day 28, representative of n=3 mice/group. (D) Immunohistochemistry staining of FBXL2 and FBXO3 in mouse isograft (left) and allograft (right) lungs at day 28. (E) Densitometry analysis of Immunohistochemical staining of FBXO3 and FBXL2 for (D). Data are representative of n=3 mice/group. (F) Immunohistochemistry staining of CD3 T cells in mouse isograft (left) and allograft (right) lungs at day 28, representative of n=3 mice/group. (G) Three explanted human lungs from normal donors or patients with end-stage CLAD/BOS were homogenized and assayed for FBXO3, FBXL2, T-bet, and IFN-γ immunoblots. (H) Densitometry for FBXO3, FBXL2, T-bet, and IFN-γ quantification from (E). Cumulative data showing densitometry of the control lungs (black columns) and BOS lungs (grey columns). All results are representative of three replicate experiments. Student t test *p < 0.05, **p < 0.005.

FBXL2 ubiquitinates and regulates T-bet

A previous study showed that T-bet undergoes poly-ubiquitination, though the factor’s leading to this process were not defined(19). Because we observed an inverse relationship between FBXL2 and T-bet in our acute and chronic lung rejection studies, we set out to test whether FBXl2 regulates T-bet. We performed an in vitro ubiquitination assay with/without recombinant FBXL2-Flag and components of E3 ligase scaffolding components. The addition of FBXL2 to the E3 ligase E1, E2, SCF, and ubiquitin resulted in a poly-ubiquitination of T-bet-HA protein in vitro (Figure 4A). We next performed immunoprecipitation pull-down experiments in the Jurkat T cell line with/without activation using anti CD3/28 antibodies previously shown to induce T-bet expression. We found that pull-down of T-bet protein co-precipitated with FBXL2 protein (Figure 4B) and pull-down of FBXL2 resulted in co-precipitation with T-bet (Figure 4C). We then used the MLE12 cell line to co-transfect with the constant level of T-bet-HA and varying concentrations of recombinant FBXL2 protein. The addition of FBXL2 resulted in diminished T-bet-HA protein level in a dose-dependent manner (Figure 4D). Together, these data provide evidence that FBXL2 protein ubiquitinates, co-precipitates with, and negatively regulates T-bet protein.

Figure 4. FBXL2 ubiquitinates and regulates T-bet.

Figure 4.

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(A) In vitro ubiquitination assay using purified E1/E2/E3 complex components were incubated with HA-T-bet and the full complement of ubiquitination reaction components (second lane from left). Data representative of 3 replicate experiments. Jurkat cells were stimulated with/without αCD3/CD28 antibodies for 24h and cells were then lysed either T-bet (B) or FBXL2 (C) was immunoprecipitated followed by FBXL2 (B) or T-bet (C) immunoblotting. Data are representative of three replicate experiments. (D) MLE12 cells were transfected with increasing amounts of FBXL2 E3 ligase plasmids with fixed amount of T-bet plasmid for 18h. The cell lysates were separated by SDS (bottom) PAGE and analyzed by immunoblotting with the indicated Abs. Data are representative of three replicate experiments.

TNF-α negatively regulates FBXL2 at the protein and mRNA levels.

Because we detected an increased FBXO3:FBXL2 protein balance in the setting of Type-1 allo-immune response in the mouse OLT model and human system, we asked whether Type-1 cytokines could impact either FBXO3 or FBXL2 expression. We therefore tested resting and adherent MLE12 cells in the presence or absence of varying doses of the Type-1 cytokines TNF-α, IL-1β and IFN-γ at 0h, 4h, 8h and 24h. The addition of IFN-γ or IL-1β had no effect on the expression of FBXO3 at the protein level (Figure 5A-F). However, the addition of recombinant TNF-α resulted in significant diminution of FBXL2 protein. We further tested exogenous TNF-α and found that it also decreased FBXL2 at the mRNA level (Figure 5G). We also compared day 10 isograft and allografts and found that FBXL2 and TNF-α mRNA levels were inversely correlated, further support a role for regulation in vivo (Figure 5H). Together, these data demonstrate that the Type 1 inflammatory cytokine, TNF-α, negatively cross-regulates FBXL2 at the protein and mRNA levels.

Figure 5. TNF-α down-regulates FBXL2 at the protein and mRNA levels.

Figure 5.

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(A-F) MLE-12 cells were cultured and incubated with murine recombinant TNF-α, IFN-γ and IL1β at 1, 10 and 100 ng for 8 hours and 10 ng for 0, 4, 8 and 24 h. Protein expression of FBXL2 in MLE-12 cells was detected by western blot assay. Data are representative of three replicate experiments. (G) MLE-12 cells were cultured and incubated with murine recombinant TNF-α at 10 and 100 ng for 4 hours. The relative mRNA expression levels of FBXL2 were measured by quantitative RT-PCR after 4 hours of treatment. All results are representative of three replicate experiments. Student t-test *p < 0.05. (H) Cumulative data showing the mRNA expression level of TNF-α and FBXL2 in isograft (grey columns) and allograft mouse lungs (black columns) at day 10, as detected by quantitative RT-PCR. All results are representative of three replicate experiments, using Student t-test *p < 0.05.

DISCUSSION

Herein, we demonstrate that the combinatorial system of E3 ligase subunits, FBXO3 and FBXL2, are altered during lung allograft rejection with potent induction of the pro-inflammatory FBXO3 and a diminution in anti-inflammatory FBXL2. This change in FBXO3:FBXL2 balance results in striking up-regulation of the TRAF proteins (TRAF1–5) that we previously have shown are regulated by FBXL2, as well as significant induction of the Type-1 immune response transcription factor, T-bet, and the hallmark effector cytokine, IFN-γ. The predominance of Type-1 alloimmune responses in this mouse OLT model during acute allograft rejection in wild-type mice has been previously demonstrated by our group and others(5, 20, 21). We also found that costimulation blockade using MR1/CTLA4-Ig, which has been previously shown to block ACR and Type-1 alloimmune responses(5, 22), results in significantly diminished FBXO3 induction, preservation of FBXL2 levels and marked reduction of the TRAFs, demonstrating that changes in the FBXO3:FBXL2 are highly dependent on alloimmune T cell-dependent inflammation. This is the first demonstration that we are aware showing the combinatorial FBXO3/FBXL2 E3 ligase system being downstream of an adaptive T cell response. Previously, we and others have shown marked FBXO3 induction in response to LPS challenge and influenza or Rift Valley fever virus infections, with degradation of FBXL2 and the p62 subunit of general transcription factor TFIIH(8, 12, 23). Targeting of FBXO3 using small molecules may be effective in attenuating lung inflammation(12, 24, 25). Consistent with this, we observed increased FBXO3 during allograft rejection along with a concomitant reduction in FBXL2 protein levels. Similarly, we found persistently increased FBXO3:FBXL2 balance in our chronic rejection model at day 28, in addition to ongoing elevated levels of T-bet and IFN-γ compared to isografts or native lungs from allograft recipients. We then found this system to be demonstrably active in human CLAD lungs, with elevated FBXO3:FBXL2 protein balance in conjunction with increased T-bet and IFN-γ levels. Together, our findings suggested a potential immunoregulatory role for this E3 ligase system, given the observed inverse correlation between FBXL2 and T-bet/IFN-γ protein levels.

Airway epithelial cells play a major role in maintaining lung mucosal integrity and host defense and are critical target during acute and chronic lung allograft rejection(26, 27). During acute and chronic rejection, we observed a marked increase in FBXO3 immunohistochemical staining in airway epithelia and peri-airway infiltrating mononuclear cells (including CD3+ T cells) compared to isografts or costimulation blockade-treated allograft lungs. In contrast, we saw reduced airway epithelial tissue staining for FBXL2 during acute and chronic rejection compared to higher levels in isografts or treated allografts. Collectively, our findings underscore the importance of both airway epithelia and infiltrating immune cells in our observed changes in FBXO3:FBXL2 balance during allograft rejection and the important role of T cell alloimmunity in regulating this combinatorial E3 ligase system. These findings led us to probe whether Type-1 cytokines directly regulated either FBXO3 or FBXL2 and indeed we found that TNF-α directly decreased FBXL2 at the mRNA and protein levels. Thus, while we previously have shown that FBXO3 degrades FBXL2, to our knowledge, this is the first demonstration of an inflammatory cytokine regulating an E3 ligase subunit. This immunoregulation is highly plausible in vivo, as we have previously shown TNF-α to be produced by allospecific T cells during airway or lung allograft rejection in both the mouse and human and can also be induced from macrophages in response to IFN-γ(5, 28, 29). Together our findings demonstrate that lung allograft rejection and its associated inflammation result in a significant change in the balance of the FBXO3:FBXL2 E3 ligase subunit system, with significant reduction in the immune checkpoint, FBXL2. Thus, we propose a revised model for the cross regulation of the combinatorial FBXO3:FBXL2 E3-ligase system with the transcription factor, T-bet, and the Type-1 T cell cytokines TNF-a and IFN-g during lung allograft rejection/tolerance (Figure 6).

Figure 6.

Figure 6.

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Cross Regulation of the combinatorial FBXO3/FBXL2 E3-ligase system with the transcription factor, T-bet, and the Type-1 T cell cytokines TNF-a and IFN-g during lung allograft rejection. The pro-inflammatory FBXO3 is induced during lung allograft rejection and ubiquitinates and degrades the anti-inflammatory FBXL2, which ubiquitinates and regulates the Type-1 transcription factor, T-bet. Release of the Type-1 cytokine, TNF-a, negatively cross regulates FBXL2.

Our previous work showed that T-bet–deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade–resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8+IL-17+ T cells(21). Other studies in the mouse heterotopic heart transplant model similarly demonstrated accelerated allograft rejection in the absence of T-bet that was mediated by IL-17-producing T cells (30, 31). Earlier human studies showed a correlation of high TNF-α plasma levels with heart, renal and liver allograft rejection (32–36). We previously reported that in both the heterotopic tracheal transplant model and the mouse orthotopic lung that allospecific CD8+ T cells produce TNF-α during acute rejection in MHC-mismatched grafts(21, 28). Recently, we demonstrated that human lung transplant recipients with CLAD have increased TNF-α mRNA and protein detected in the airway transcriptome and bronchoalveolar lavage fluid, respectively (6). Together these studies suggest important roles for both T-bet and TNF-α in allograft rejection.

CLAD continues to be the major barrier to long-term survival in LTRs and to date, there remains a paucity of biomarkers associated with disease. We recently evaluated the small airway transcriptome in BOS/CLAD LTRs compared to controls and found a signature in CLAD marking a predominant Type-1 immune response(37). Interestingly TNF-α was up-regulated in CLAD at the mRNA and protein levels, along with donor-specific CD4+ and CD8+ alloimmune responses. Further, TNF-α was identified as a major upstream regulator of the airway transcriptome. Thus, our data in BOS/CLAD lungs showing significant downregulation of FBXL2 protein is consistent with these immune findings in CLAD and could suggest TNF-α as a potential therapeutic target in CLAD, though this needs to be balanced with increased infectious risk(38). In contrast, therapies that promote FBXL2 preservation might also be considered as a therapeutic target for lung allograft acceptance.

A previous report demonstrated that T-bet is polyubiquitinated at lysine sites leading to decreased protein stability, however the factor(s) leading to ubiquitination were not elucidated(19). In contrast, USP10 has been shown to deubiquitinate and stabilize T-bet(39). Because we found T-bet was potently induced and remained elevated from acute into chronic lung allograft rejection and correlated with diminished FBXL2 levels, we tested the hypothesis that FBXL2 was an E3 ligase subunit regulator of T-bet. Using the Jurkat T cell line, a system previously shown to have activation-dependent T-bet expression(40, 41), we demonstrate that both T-bet and FBXL2 co-precipitate with each other following anti-CD3/anti-CD28 stimulation. In addition, we found that exogenous FBXL2 protein, in the presence of other SCF components, was sufficient to polyubiquitinate T-bet. Moreover, we found that FBXL2 decreased T-bet stability in a dose-dependent manner. To our knowledge, this is the first factor that we are aware, that has been shown to ubiquitinate T-bet. Thus, our data suggest that diminished FBXL2 during lung allograft rejection may contribute to an ongoing Type-1 immune response via T-bet stabilization.

In conclusion, we find that the E3 ligase combinatorial FBXO3:FBXL2 balance is significantly increased during acute and chronic lung allograft rejection in the mouse OLT model and human BOS/CLAD lungs. Reduced FBXL2 expression occurs in the context of a robust Type-1 alloimmune response, with TNF-α shown to be an additional negative regulator of this important immune checkpoint. Cross regulation of T-bet, by FBXL2 was also found, demonstrating an important mechanism for immunoregulation of Type-1 inflammation that could possibly be exploited to mitigate allograft rejection.

Supplementary Material

1

Key points:

FBXO3:FBXL2 balance is significantly increased during lung allograft rejection.

TNF-α negatively regulates FBXL2 expression.

FBxl2 ubiquitinates and regulates T-bet expression.

Funding:

This work was supported by National Institutes of Health grants R01HL133184 (JFM, BBC) and P01HL114453 and R01HL081784 (RKM)

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