Maintenance of IKKβ activity is necessary to protect lung grafts from acute injury

Abstract

Background: Signaling pathways that target I-κB kinase β (IKKβ) activation stimulate the expression of nuclear factor (NF)-κB-dependent genes and are thus believed to primarily promote inflammation and injury in solid organ grafts.

Methods: We examined the role of IKKβ in a mouse model of lung transplantation-mediated ischemia-reperfusion injury using NF-κB essential modulator (NEMO)-binding domain (NBD) peptide to pharmacologically inhibit IKK activation. As myeloid cells are primarily responsible for the production of acute inflammatory mediators after lung transplantation, we also investigated the effects of myeloid cell-specific IKKβ gene deletion on acute lung graft injury by transplanting mutant mice.

Results: When NBD was administered at a dose that partially inhibits IKKβ activation, we observed attenuated lung graft injury and blunted expression of intragraft proinflammatory mediators. Surprisingly, when the dose of NBD was increased to a level that ablates intragraft IKKβ activation, graft inflammation, and injury were significantly worse compared with recipients treated with control peptide. Similar to lung recipients with pharmacologically ablated IKKβ activity, donor-recipient transplant combinations with a myeloid cell-specific IKKβ gene deletion had marked intragraft inflammation and poor lung function.

Conclusions: Our data show maintenance of IKKβ activity is critical for promoting graft homeostasis with important implications for targeting NF-κB-dependent signaling pathways for treating acute lung injury.

Figure 1

The effects of pharmacological blockade of IKKβ on lung graft injury. (a) Sham-operated mice or B6 → B6 lung recipients treated with NBD-C, NBD^low or NBD^hi and assessed for (left panel) PaO₂ (N=5) or (right panel) EBD dye exclusion (N=5) 24 hours after engraftment. Data are shown as the mean ± standard error of the mean (SEM) *, p<0.05. (b) Representative (N=5) graft histology (200x magnification) and a (c) representative (N=5) graft tissue TUNEL assay (200x magnification) from indicated lung recipients 24 hrs following transplantation. (d) Measurement of serum creatine phosphokinase (sCPK), alanine aminotransferase (sALT) and creatinine (sCr) in NBD^hi or NBD-C-treated B6 → B6 lung recipients (N=4) 24 hours after engraftment. Data are shown as the mean ± SEM. (e) Representative FACS analysis (N=5) of percent abundance of intragraft granulocytes in B6 → B6 lung recipients treated with NBD-C, NBD^low or NBD^hi 24 hours after engraftment.

Figure 2

The dynamics of intragraft IKKβ activity and inflammatory gene expression following pharmacological blockade. (a) B6 → B6 lung recipients were treated with NBD-C, NBD^low or NBD^hi and evaluated for IKKβ activity at indicated times. Results are representative of 2 independent experiments. Results shown are from one representative experiment where N ≥ 4 per time point and is normalized to IKKβ activity of B6 lung tissue following a sham operation. (b) B6 → B6 lung recipients were treated as in (a) and analyzed for levels of intragraft inflammatory mediator transcripts by real-time quantitative RT-PCR at indicated times. Results shown as a mean ± SEM *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 3

Graft injury in IKKβ^Δmye lung recipients. (a) IKKβ^fl/fl or IKKβ^Δmye mice underwent sham operations or IKKβ^fl/fl → IKKβ^fl/fl, IKKβ^Δmye → IKKβ^fl/fl, IKKβ^fl/fl → IKKβ^Δmye, IKKβ^Δmye → IKKβ^Δmye lung transplants were performed and evaluated for graft (left panel) PaO₂ (N=4) or (right panel) Evans Blue dye (EBD) exclusion (N=4) 24 hours after engraftment. Results are shown as a mean ± SEM *, p < 0.05, n.s, p = 0.14 (b) Representative (N=5) histopathological analysis of indicated lung grafts 24 hours after transplantation (100x magnification, inset 400x magnification).

Figure 4

Accumulation of granulocytes in IKKβ^Δmye lung recipients. IKKβ^fl/fl → IKKβ^fl/fl and IKKβ^Δmye → IKKβ^Δmye lung transplants were performed and assessed for intragraft granulocyte accumulation. (a) Representative FACS analysis (N=5) of the percent abundance of granulocytes in lung graft tissue, (b) total granulocyte counts in the (upper panel) airway and (lower panel) lung graft tissue 24 hours after engraftment. (c) Representative TUNEL assay (N=3) on graft tissue from IKKβ^fl/fl → IKKβ^fl/fl and IKKβ^Δmye → IKKβ^Δmye lung recipients 24 hrs following transplantation (200x magnification). (d) Representative histological analysis (N=5) of control IgG or Ly6G antibody-treated IKKβ^Δmye → IKKβ^Δmye lung recipients (200x magnification, inset 400x magnification). (e) IKKβ^Δmye → IKKβ^Δmye lung recipients (N=5) treated as in (d) and assessed for PaO₂. For (b) and (d) results are shown as a mean ± SEM *, p < 0.05.

Figure 5

Inflammatory mediator expression in IKKβ^Δmye lung recipients. IKKβ^fl/fl → IKKβ^fl/fl (N=4) and IKKβ^Δmye → IKKβ^Δmye (N=4) lung transplants were performed and compared for intragraft inflammatory mediator expression by multiplex ELISA at 24 hours following engraftment. Results are shown as a mean ± SEM *, p < 0.05.