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.

Abstract

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-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 with 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 with 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 lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial 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.

Footnotes

This work was supported by Foundation for the National Institutes of Health Grants R01HL133184 (to J.F.M. and B.B.C.) and P01HL114453 and R01HL081784 (to R.K.M.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

ACRacute cellular rejectionBOSbronchiolitis obliterans syndromeCLADchronic lung allograft dysfunctionFBXL2F-box and leucine-rich repeat protein 2FBXO3F-box protein 3HAhemagglutininIHCimmunohistochemistryLTRlung transplant recipientMLEmouse lung epithelialOLTorthotopic lung transplantSCFSkip-Cullin1–F-box proteinTRAFTNFR-associated factorReceived April 5, 2022.Accepted August 15, 2022.Copyright © 2022 by The American Association of Immunologists, Inc.