Pin1 function on type 1 cytokine and chemokine expression was first evaluated in Pin1 knockout (KO) mice. Splenocytes from KO mice activated with anti-CD3 plus anti-CD28, which normally triggers cytokine mRNA stabilization and accumulation 
, showed significantly less IFN-γ and IL-2 mRNA compared to WT (p<0.03 and p<0.008, respectively) while CXCL-10 mRNA was reduced by 50% but did not quite reach significance (). Secreted IFN-γ was proportionally reduced (4-fold) in the supernatant of KO splenocyte cultures compared to WT (). Bulk analysis of activated KO CD4+ or CD8+ splenocytes by flow cytometry showed reductions in IL-2 and IFN-γ positive cells () compared to splenocytes from heterozygote mice. In KO mice, no differences were noted in the numbers of splenic or thymic CD3, CD4, CD8 or regulatory T cell populations or activation marker expression after stimulation (not shown
), eliminating developmental differences between WT and KO mice. As CD3 mediated signaling is necessary for T cell development, these data suggest TCR function is likely normal in Pin1 KO animals. Instead, these data suggested Pin1 was involved in co-stimulatory-CD3/CD28 signaling. Indeed, IFN-γ and IL-2 mRNAs were less stable in anti-CD3/anti-CD28 activated KO than WT splenocytes while the stability of CXCL-10 mRNA, which lacks AREs was unchanged ( and not shown
). Therefore, Pin1 is necessary for ARE mediated cytokine mRNA stabilization after T cell co-stimulation. As Pin1 substrates also include NF-κB and NF-AT 
, which regulate cytokine mRNA transcription, the observed reductions in CXCL-10 suggest a nuclear event.
Figure 1 Cytokine expression is downregulated in Pin1−/− mouse. 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 (more ...)
Pin1 can be selectively blocked by juglone (5-hydroxy-1,4-naphthoquinone), which covalently binds to and irreversibly inhibits Pin-1 activity but has no effect on CyA or FKBP 
. Prior to its use in vivo
, WT and KO thymocytes were activated with anti-CD3/anti-CD28 while rat splenocytes were treated with ionophore/PMA (I/P) (, and not shown).
After 4 hours of I/P, cytokine mRNAs increased by as much as 500 fold (IFN-γ. IFN-γ and IL-2 mRNA levels were reduced in the rat or mouse WT cells by 70–95% after treatment with juglone (,
). However, juglone had no significant effect on the abundance of IL-2, IFN-γ or the housekeeping RNAs in Pin1 KO cells ( and not shown
), demonstrating the specificity of this drug. At the concentrations used here neither CyA nor FKBP12 peptidyl-prolyl isomerase activities are affected 
. Juglone (1 µM) completely blocked the accumulation of I/P-induced IFN-γ and IL-2 mRNAs by rat splenocytes without altering actin (). At lower juglone concentrations (0.1 µM), only the accumulation of IFN-γ mRNA was significantly inhibited (). These data suggest a variable sensitivity of cytokine mRNAs to Pin1 inhibition. The accumulation of IL-2 and IFN-γ after juglone were dramatically reduced in the culture supernatants (). The failure to produce cytokines was not a function of cell death as equivalent levels of viability were observed in I/P versus I/P/juglone treated splenocytes ().
Figure 2 Cytokine mRNAs are suppressed by Pin1 inhibition in activated rat splenocytes. 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 (more ...)
In order to characterize Pin1 function during an in vivo
type I immune response, we used the widely employed F344 to WKY rat, MHC Class I mismatched, orthotopic, single lung transplantation model 
. The donor organ is attached via cuffs to the recipient's bronchial and vascular systems permitting normal function. Nonimmunosuppressed recipients experience profound acute rejection within several days largely mediated by IFN-γ and CXCL-10 upregulation 
. Over several weeks, chronic rejection occurs with alveolar, pleural, and peribronchial collagen deposition, loss of viable pneumocytes and eventual organ loss. Recipients were given a daily, single intraperitoneal (IP) injection of 1 mg/kg juglone dissolved in ethanol and diluted in 5 ml saline, while controls received diluents only. This doses of juglone had no effect on red cell mass, white cell counts, serum chemistries, liver function tests, or renal function in control, untransplanted rats (not shown)
. Therefore juglone administration appeared to be safe and nontoxic. Treatment was started the day of the transplant. At day 7 or 14, animals were sacrificed and the lungs evaluated grossly and by histopathology. In untreated animals, the transplant was visibly shrunken and the pleural surfaces hemorrhagic (). Palpation revealed a firm and unyielding consistency. The juglone treated animals showed no gross signs of rejection () and was indistinguishable from the contralateral control. Microscopically, the untreated transplanted lung showed severe rejection with acute inflammatory cell infiltration predominantly composed of neutrophils, lymphocytes and macrophages. Alveolar architecture was totally effaced and the small airways packed with inflammatory cells (). These changes were completely absent in juglone treated animals, which showed normal alveolar architecture, pleural thickness, and airway patency (). Occasional round macrophages were present in some alveoli. No significant difference was found in the relative proportion of CD4, CD8 or γδ T cells in blood and BAL fluid from the native right lung or the transplanted left lung, 7 days after transplantation in juglone treated or untreated animals (not shown and )
. This is in contrast to collagen V tolerized animals that show predominantly CD4+
T cells 
. These data demonstrate that Pin1 blockade can dramatically attenuate acute transplant rejection.
Figure 3 Pin1 is required for acute and chronic rejection. 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 (more ...)
Figure 4 IFN-γ and IL-2 are Pin1 dependent. 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 (more ...)
One week after transplantation the levels of IFN-γ, and IL-2 in the bronchoalveolar fluid (BAL) were reduced (). These data suggest that Pin1 blockade prevented cytokine production by T cells, which were the majority population within the BAL fluid (not shown
). Since draining lymph nodes are a predominant site for immunological reactivity we analyzed mediastinal lymph node cells for the expression of cytokine mRNAs by qPCR. mRNAs coding for IFN-γ, IL-2, and CXCL-10 were significantly lower in juglone treated animals than controls whereas IL-4 was not significantly affected (). IFN-γ ELISPOTS of whole spleen showed large reductions in the juglone treated animals from untreated controls (). At day 7, Pin1 PPIase activity and amount were attenuated in BAL cells and draining lymph nodes in juglone treated rats (). Once inactivated by juglone, Pin1 is rapidly degraded by the proteasome 
. Therefore, Pin1 inhibition prevented IFN-γ, and IL-2 expression by T cells throughout the immune system and acute graft rejection after lung transplantation. The profound suppression of CXCL-10 presumably reflects both reductions in IFN-γ, which normally induces CXCL-10 as well as direct effects of Pin1 on CXCL-10 expression.
Figure 5 Expression of exogenous IFN-γ and CXCL-10 in juglone-treated rats overcomes Pin1 inhibition and results in rejection. A/ BAL fluid cells from transplanted and untreated controls (), or juglone treated () were quantitated, lysed (more ...)
The expression of these cytokines has been highly correlated with rejection in both animal models and humans 
. In order to demonstrate a causal role, IFN-γ and CXCL-10 expression vectors were combined and insufflated into donor lungs immediately before religature in the recipient. To avoid potential Pin1 mediated, post-transcriptional regulation, only the coding region without the 3′ UTR was inserted downstream of a constitutively active CMV promoter. By 1 week after transplant, the lung grafts underwent significant rejection in untreated controls, which was prevented by juglone (). The forced expression of IFN-γ and CXCL-10 in juglone treated rats resulted in severe cellular infiltration irrespective of Pin1 blockade (). Transgenic cytokines were detectable in the BAL at comparable levels to that seen in untreated recipients (not shown
). These results support a central role of IFN-γ and CXCL-10 in the process of acute rejection and suggest that cytokine suppression after Pin1 inhibition is likely responsible for graft sparing.
Immunosuppressive drugs, particularly CsA greatly improves graft survival but have renal damage as a common side effect 
. To avoid toxicity and enhance graft acceptance, immunosuppressants such as corticosteroids, calcineurin inhibitors, antimetabolites, and rapamycin are given in combination 
. Cyclosporine A and tacrolimus indirectly inhibit nuclear factor of T cells (NFAT) preventing the transcriptional upregulation of cytokine genes by activated T cells 
. In contrast, Pin1 suppresses expression through the regulation of cytokine mRNA stability. Given this distinct mode of action, we asked if combined CsA and juglone therapy would be additive or synergistic. CsA was injected intraperitoneally for 3 days at a dose of 1 mg/kg together with 0.1 mg/kg of juglone, which was then continued alone for 4 more days. Cyclosporine is usually used at 25 mg/kg/day for 3 days which induces long lived lung transplant acceptance in rats while moderate to severe rejection was observed at doses of 5 mg/kg/d 
. Transplanted animals treated with CsA alone (1 mg/kg/d) showed modest graft discoloration () but microscopically severe cellular infiltration diffusely throughout the parenchyma with foci in peribronchial and perivascular areas (). Similarly, suboptimal inhibition of Pin1 leads to severe rejection pathology with substantial cellular infiltration and hemorrhage (). In contrast, combined suboptimal treatment with CsA and juglone provided excellent graft protection without identifiable cellular infiltrates (). These data show that combined inhibition of Pin1 and calcineurin are additive or synergistic and suggest that CsA dosage could be signifcantly reduced if Pin1 inhibition is added to the therapeutic regiment.
Figure 6 Pin1 and calcineurin inhibition are synergistic. Transplants were performed as for 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 (more ...)