CD26 as a target against fibrous formation in chronic airway rejection lesions.

AIMS: Chronic lung allograft dysfunction (CLAD) after lung transplantation (Tx) is the clinical result of chronic airway rejection lesions (CARL), histomorphologically described as either obliterative remodeling of small airways or alveolar fibroelastosis, or as a combination of both. We here investigated the CD26-inhibitory effect on CD26-expressing CARL. MAIN METHODS: CARL were induced by BALB/c â†’ C57BL/6 mouse Tx under mild immunosuppression. CARL-related pro-fibrotic mediators were determined by RT-qPCR and western blotting (WB), EMT and ERK markers by WB. CD26 co-expression by immunofluorescence. CD26 was inhibited by Vildagliptin, gene depleted by CD26-/- mice. Primary lung fibroblasts were employed for ex vivo analyses. Samples from lung transplant patients with CLAD were analyzed by immunohistochemistry. KEY FINDINGS: CARL revealed a significantly higher expression of profibrotic proteins vs. normal lungs (p < 0.05). CD26 and EMT co-expressed in CARL with significantly higher Vimentin, Slug, Hif-1α, α-SMA expression vs. normal lungs (p < 0.05). Vildagliptin decreased the expression of α-SMA and N-cadherin in wild type (WT) lung fibroblasts (p < 0.05). Primary lung fibroblasts from WT and CD26-/- mice treated with TGF-ß1, IFN-γ, and FGF showed a reduction of EMT protein expression, proliferation, and reduced activation of ERK in CD26-/- mice vs. WT mice. CD26-positive cells were found in patient samples with CLAD in areas of loose fibrosis, but not in areas of dense fibrosis. SIGNIFICANCE: CD26 is expressed in CARL-developing lung transplants and CD26-inhibition downregulates fibrosis-forming mediators and fibroblast proliferation. CD26 thus qualifies as a target to attenuate the development of CARL mainly via modulation of ERK and the EMT pathway.

The Amide Local Anesthetic Ropivacaine Attenuates Acute Rejection After Allogeneic Mouse Lung Transplantation.

PURPOSE: Acute allograft rejection after lung transplantation remains an unsolved hurdle. The pathogenesis includes an inflammatory response during and after transplantation. Ropivacaine, an amide-linked local anesthetic, has been shown to attenuate lung injury due to its anti-inflammatory effects. We hypothesized that the drug would also be able to attenuate acute rejection (AR) after allogeneic lung transplantation. METHODS: Allogeneic, orthotopic, single left lung transplantation was performed between BALB/c (donors) and C57BL/6 (recipients) mice. Prior to explantation, lungs were flushed with normal saline with or without ropivacaine (final concentration 1 µM). Plasma levels of tumor necrosis factor-α and interleukins - 6 and - 10 were measured 3 h after transplantation by ELISA. Lung function was assessed on postoperative day five and transplanted lungs were analyzed using histology (AR), immunohistochemistry (infiltrating leukocytes) and Western blot (phosphorylation and expression of Src and caveolin-1). RESULTS: Ropivacaine pre-treatment significantly reduced AR scores (median 3 [minimum-maximum 2-4] for control vs. 2 [1-2] for ropivacaine, p < 0.001) and plasma levels of tumor necrosis factor-α (p = 0.01) compared to control, whereas plasma concentrations of interleukin - 6 (p = 0.008) and - 10 (p < 0.001) were increased by ropivacaine. The number of T-lymphocytes infiltrating the transplanted lung was attenuated (p = 0.02), while no differences in macrophage or B-lymphocyte numbers could be observed after ropivacaine pre-treatment. Caveolin-1 phosphorylation in ropivacaine-treated lungs was diminished (p = 0.004). CONCLUSIONS: Pre-treatment of donor lungs with the local anesthetic ropivacaine diminished histological signs of AR after orthotopic left lung transplantation in mice, most likely due to reduced infiltration of T-lymphocytes into the graft.

The CD26/DPP4-inhibitor vildagliptin suppresses lung cancer growth via macrophage-mediated NK cell activity.

CD26/dipeptidyl peptidase 4 (DPP4) is a transmembrane protein which is expressed by various malignant cells. We found that the expression of CD26/DPP4 was significantly higher in lung adenocarcinoma samples in our own patient cohort compared to normal lung tissue. We therefore hypothesize that the inhibition of CD26/DPP4 can potentially suppress lung cancer growth. The CD26/DPP4 inhibitor vildagliptin was employed on Lewis Lung Carcinoma (LLC) cell line and a human lung adenocarcinoma (H460) cell line. Two weeks after subcutaneous injection of tumor cells into C57BL/6 and CD1/nude mice, the size of LLC and H460 tumors was significantly reduced by vildagliptin. Immunohistochemically, the number of macrophages (F4/80+) and NK cells (NKp46+) was significantly increased in vildagliptin-treated tumor samples. Mechanistically, we found in vitro that lung cancer cell lines expressed increased levels of surfactant protein upon vildagliptin treatment thereby promoting the pro-inflammatory activity of macrophages. By the depletion of macrophages with clodronate and by using NK cell deficient (IL-15-/-) mice, tumors reversed to the size of controls, suggesting that indeed macrophages and NK cells were responsible for the observed tumor-suppressing effect upon vildagliptin treatment. FACS analysis showed tumor-infiltrating NK cells to express tumor necrosis-related apoptosis-inducing ligand (TRAIL) which induced the intra-cellular stress marker γH2AX. Accordingly, we found upregulated γH2AX in vildagliptin-treated tumors and TRAIL-treated cell lines. Moreover, the effect of vildagliptin-mediated enhanced NK cell cytotoxicity could be reversed by antagonizing the TRAIL receptor. Our data provide evidence that the CD26/DPP4-inhibitor vildagliptin reduces lung cancer growth. We could demonstrate that this effect is exerted by surfactant-activated macrophages and NK cells that act against the tumor via TRAIL-mediated cytotoxicity.

Preclinical, non-genetic models of lung adenocarcinoma: a comparative survey.

Lung cancer is the leading cause of cancer-related mortality worldwide. Animal models are key in analyzing cancer biology and therapy evaluation. We here compared relevant non-genetic lung cancer models with regard to tumor induction period, incidence, morbidity and mortality rate and the immunological composition of primary tumors and the occurrence of tertiary lymphoid organs (TLO): (I) intraperitoneal Urethane injection (1g/kg), (II) Lewis lung carcinoma (LLC) cell line model (intravenous or subcutaneous), and (III) ex vivo three-dimensional (3D) primary cell culture model established from subcutaneously developed LLC-induced tumors. The incidence of Urethane induced lung tumors was 100% in both, C57BL/6 and BALB/c strains without morbidity or mortality at twenty weeks after injection. The mean size of tumor nodules after Urethane injection was significantly larger in BALB/c mice vs. C57BL/6 (p<0.01). Three times of Urethane injection produced significantly more tumor nodules in both mouse strains compared to one injection (BALB/c: p<0.01; C57BL/6: p<0.05). TLOs were only found in the Urethane-induced model. Although the cell line models also showed 100% induction rate, morbidity was high due to skin ulceration on the inoculation site and the development of pleural effusions in the subcutaneous model and the intravenous model, respectively. Tendencies, but no significant differences (p>0.05) could be found in the count of CD4+, CD8+, F4/80+ and NKp46+ cells in a tumor nodule among investigated models. All discussed models provided a high tumor incidence rate. TLOs were exclusively found in the Urethane-induced model. No significant difference could be found regarding immune cells across models.

Anti-inflammatory effects on ischemia/reperfusion-injured lung transplants by the cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) inhibitor vildagliptin.

OBJECTIVES: We showed previously that stromal cell-derived factor 1 (SDF-1) is a substrate of cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) and exerts regenerative properties on acute lung ischemia-reperfusion injury on CD26/DPP4 inhibition. Here, we extend our studies to test whether an intermediate recovery of lung transplants from ischemia/reperfusion injury by CD26/DPP4 inhibition can be achieved for up to 14 days. METHODS: Syngeneic mouse lung transplantation (Tx) was performed in C57BL/6 and in CD26-/- mice by applying 18 hours of cold ischemia. Donor lungs were preconditioned with saline or the CD26/DPP4 inhibitor vildagliptin (1 µg/mL [3 µM]). In vitro, the influence of vildagliptin and SDF-1 on the macrophage cell line RAW 264.7 was tested. Transplants were analyzed up to 14 days after Tx for the expression of SDF-1, tumor necrosis factor-α (TNF-α), interleukin-10, intercellular adhesion molecule-1 (ICAM-1), immune cell infiltration, and oxygenation. RESULTS: Cold ischemic time of 18 hours with vildagliptin preconditioning elevated lung SDF-1 levels (P = .0011) and increased interleukin-10-producing macrophages (P = .0165) compared with the control. SDF-1 reduced macrophage-derived TNF-α (P = .0248) in vitro. Five hours after Tx, vildagliptin significantly reduced macrophages and neutrophils (P = .0306), decreased ICAM-1 expression (P = .002), and improved transplant oxygenation (P = .0181). Seven days after Tx, grafts were preserved on CD26/DPP4-inhibition: perivascular macrophages (P = .0046) and TNF-α (P = .0013) were reduced as well as T and B cells. ICAM-1 was absent in CD26/DPP4-inhibited grafts at all time points. CONCLUSIONS: This study proves an intermediate improvement of ischemia/reperfusion-injured lung transplants by the CD26/DPP4-inhibitor vildagliptin up to 14 days. Enhanced levels of SDF-1 induced an anti-inflammatory effect on a cellular and protein level, and render CD26/DPP4 inhibition preconditioning effective for the protection from lung ischemia/reperfusion injury.