Pin1 Modulates the Type 1 Immune Response

BACKGROUND/ABSTRACT: Immune responses initiated by T cell receptor (TCR) and costimulatory molecule mediated signaling culminate in maximal cytokine mRNA production and stability. The transcriptional responses to co-stimulatory T cell signalling involve calcineurin and NF-AT, which can be antagonized by interference with the cis-trans peptidyl-prolyl isomerases (PPIase), cyclophilin A and FKBP. Signalling molecules downstream of CD28 which are essential for the stabilization of cytokine mRNAs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We now show that Pin1, a third member of the PPIase family mediates the post-transcriptional regulation of Th1 cytokines by activated T cells. Blockade of Pin1 by pharmacologic or genetic means greatly attenuated IFN-γ, IL-2 and CXCL-10 mRNA stability, accumulation and protein expression after cell activation. In vivo, Pin1 blockade prevented both the acute and chronic rejection of MHC mismatched, orthotopic rat lung transplants by reducing the expression of IFN-γ and CXCL-10. Combined transcriptional and post-transcriptional blockade with cyclosporine A and the Pin1 inhibitor, juglone, was synergistic. CONCLUSIONS/SIGNIFICANCE: These data suggest Pin1 inhibitors should be explored for use as immunosuppressants and employed with available calcineurin inhibitors to reduce toxicity and enhance effectiveness

Neutrophil necrosis and annexin 1 degradation associated with airway inflammation in lung transplant recipients with cystic fibrosis

<p>Abstract</p> <p>Background</p> <p>Neutrophils sequestered in lower respiratory tract secretions in the inflamed lung may undergo apoptosis and/or necrosis and release toxic cellular contents that can injure airways or parenchyma. This study examined the viability of neutrophils retrieved from the proximal airways of lung transplant recipients with bacterial tracheobronchitis.</p> <p>Methods</p> <p>Integrity and stability of intracellular proteins in neutrophils from proximal airways and peripheral blood from lung transplant recipients with bacterial tracheobronchitis were analyzed via Western blot analysis and determination of neutrophil viability by morphologic appearance and flow cytometry.</p> <p>Results</p> <p>Neutrophils in tracheobronchial secretions from lung transplant recipients with cystic fibrosis who had normal chest radiographic imaging but bronchoscopic evidence of purulent tracheobronchitis post-transplant were necrotic and associated with degradation of intracellular protein annexin 1. The neutrophil influx was compartmentalized to large airways and not detected in peripheral bronchoalveolar airspaces sampled via bronchoalveolar lavage. Peripheral blood neutrophils from healthy subjects cultured <it>in vitro</it> demonstrated that annexin 1 degradation, particularly to a 33 kDa annexin 1 breakdown product (A1-BP), was associated with neutrophil necrosis, but not apoptosis. Although annexin 1 degradation was not specific to neutrophil necrosis, it was a sensitive marker of intracellular protein degradation associated with neutrophil necrosis. Annexin 1 degradation to 33 kDa A1-BP was not observed in peripheral blood neutrophils from healthy subjects, but annexin 1 appeared to be degraded in peripheral blood neutrophils of lung transplant recipients despite a normal morphologic appearance of these cells.</p> <p>Conclusions</p> <p>Neutrophils were necrotic from the proximal airways of lung transplant recipients with bacterial tracheobronchitis, and this process may begin when neutrophils are still in the systemic circulation prior to sequestration in inflamed airways. Annexin 1 degradation to 33 kDa A1-BP may be useful as a sensitive marker to detect neutrophil necrosis.</p

Role of lung inflammatory mediators as a cause of exercise-induced arterial hypoxemia in young athletes

Effet d'un traitement pharmacologique de l'hypoxémie artérielle d'effort chez de jeunes sportifs d'endurance. Administration de fexofénadine, zileuton, nédocromil sodium. Analyse de la fonction pulmonaire, des gaz sanguins artériels, des métabolites associés au mécanisme inflammatoire : histamine, leucotriènes, prostaglandines

Figure 3

<p><i>Pin1 is required for acute and chronic rejection.</i> I and IV: Gross appearance of lungs from control, untreated transplants (I) and juglone treated (1 mg/kg) (IV). Lungs are oriented so that transplant is on the left. II and V: Hematoxylin and eosin stained sections from control untreated (II) or juglone treated (V) 1 week after transplant. III and VI: Trichrome stained sections of control, untreated (III) and juglone treated (VI) two weeks after transplant. These are representative sections from 8 control and 8 juglone treated transplant recipients in each set.</p

Figure 6

<p><i>Pin1 and calcineurin inhibition are synergistic.</i> Transplants were performed as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000226#pone-0000226-g003" target="_blank">figure 3</a> and animals harvested 7 days later. (I) Treated with CsA at 1 mg/kg/d for 3 days. (II) Treated with juglone at 0.1 mg/kg/d for 7 days. (III) Treated with CsA at 1 mg/kg/d for 3 days plus juglone at 0.1 mg/kg/d for 7 days. For all conditions, gross apearance of the transplant at harvest shown along the left, 5× and 20× representative sections stained with H & E shown in the middle and right panels.</p

Figure 2

<p><i>Cytokine mRNAs are suppressed by Pin1 inhibition in activated rat splenocytes.</i> A/ mRNAs for IFN-γ, IL-2, and CXCL-10 were analyzed in splenocytes by reverse transcription, qPCR. Cells were cultured for 4 hours without activation (resting), or with ionophore plus PMA (I/P) or I/P plus juglone (I/P/J) at 1 or 0.1 µM. The ionomycin/PMA stimulated sample was normalized to 100 and others expressed as a % of that value. The data are averages±SEM of 3 independent experiments using splenocytes of untreated healthy animals. B/ Secreted IFN-γ and IL-2 after 24 hours from the cultures as described in (A). The data are averages±SEM of 3 independent experiments using splenocytes of untreated healthy animals. C/ Viability of rat splenocytes treated as above for 24 hr, incubated with propidium iodine and analyzed by flow cytometry.</p

Figure 4

<p><i>IFN-γ and IL-2 are Pin1 dependent.</i> A/ Relative percentage of CD4, CD8 and γδ T lymphocytes in BAL cells from the transplanted lung. BAL cells were prepared from control and juglone-treated rats as described above and the percentage of CD4+, CD8+ and γδ T lymphocytes were determined by flow cytometry with specific antibodies. The graph represents an average +/− SD of 4 experiments. B/ IFN-γ and IL-2 concentrations in concentrated BAL fluid were determined by ELISA. * denotes p<0.01. C/ Mediastinal lymph nodes from control or juglone treated animals were used for reverse transcription, qPCR for the cytokines shown along the x-axis. The values in untreated controls were normalized to 100% and data shown represent % change in juglone treated samples. In (B) and (C), the data shown are an average±SEM of 4 rats in each group 7 days after transplantation. * denotes p<0.05 from control samples. D/ Splenocytes were isolated and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000226#s4" target="_blank">Material and Methods</a>. IFN-γ positive splenocytes were quantitated by Elispot (n = 3). * denotes p<0.05.</p

Figure 5

<p><i>Expression of exogenous IFN-γ and CXCL-10 in juglone-treated rats overcomes Pin1 inhibition and results in rejection</i>. A/ BAL fluid cells from transplanted and untreated controls (⧫), or juglone treated (▴) were quantitated, lysed and used for Pin1 isomerase assay. Juglone (1 µM) was also added <i>in vitro</i> to equal amounts of the control (⋄) and juglone treated (▵) samples and isomerase assay repeated. B/ Mediastinal lymph nodes from 2 untreated controls (⧫,⋄) or one juglone treated animal (▴) were lysed and equal amounts of protein used for Pin1 isomerase assay. C/ Immunoblot with anti-Pin1 or anti-actin of mediastinal lymph nodes from 2 controls, untreated (−juglone) or 2 juglone treated animals (+juglone). D/ Donor lungs were insufflated with 100 µg each of CXCL10 and IFN-γexpression vectors immediately prior to religation in recipient. Animals were then untreated (I, II) or treated with juglone at 1mg/kg for 1 week (III, IV) and histopathologic analysis after H & E staining. Representative sections from multiple animals shown.</p

Figure 1

<p><i>Cytokine expression is downregulated in Pin1−/− mouse</i>. A/ Splenocytes from Pin1−/−mice (KO) or wild-type mice (WT) were activated for 3 hours with anti-CD3 plus anti-CD28. mRNAs for IFN-γ, IL-2 or CXCL10 were analyzed by reverse transcription, qPCR. Fold change increase in activated compared to resting cells were assessed and averages±SD from 3 mice in each groups are presented. B/ Splenocytes were activated as in (A) for 5 hours and IFN-γ amounts in the cultures were measured by ELISA. Averages±SD from 3 mice in each groups are presented. C/ Percentage of IFN-γ and IL-2 positive CD4+ and CD8+ cells in splenocytes activated for 5 hours with anti-CD3. Splenocytes were incubated for 30 min at 4°C with relevant antibodies at saturation and then washed and analyzed using a FACSCalibur instrument (Becton Dickinson, Mountain View, CA). KO mice (−/−) are compared to heterozygote mice (+/−) and a representative experiment is shown D/ Splenocytes from the same mice (n = 3) were activated with anti-CD3 plus anti-CD28 for 3 hours before treated with actinomycin D (Act D;10 µg/ml). mRNA levels were determined by qPCR at the indicated times after Act D. IFN-γ mRNA levels at T₀ were normalized to 100%. E/ Thymocytes from Pin1−/− (KO) or wild-type (WT) mice were activated with anti-CD3 plus anti-CD28 for 3 hours with or without juglone (1 µM). mRNA levels were determined by qPCR and presented as relative (%) expression of IFN-γ in juglone-treated cells compared to activated. * denotes p<0.05.</p