Inhibition of breast cancer cell migration by activation of cAMP signaling.

Almost all deaths from breast cancer arise from metastasis of the transformed cells to other sites in the body. Hence, uncovering a means of inhibiting breast cancer cell migration would provide a significant advance in the treatment of this disease. Stimulation of the cAMP signaling pathway has been shown to inhibit migration and motility of a number of cell types. A very effective way of selectively stimulating cAMP signaling is through inhibition of cyclic nucleotide phosphodiesterases (PDEs). Therefore, we examined full expression profiles of all known PDE genes at the mRNA and protein levels in four human breast cancer cell lines and eight patients' breast cancer tissues. By these analyses, expression of almost all PDE genes was seen in both cell lines and tissues. In the cell lines, appreciable expression was seen for PDEs 1C, 2A, 3B, 4A, 4B, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 9A, 10A, and 11A. In patients' tissues, appreciable expression was seen for PDEs 1A, 3B, 4A, 4B, 4C, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 8B, and 9A. PDE8A mRNA in particular is prominently expressed in all cell lines and patients' tissue samples examined. We show here that stimulation of cAMP signaling with cAMP analogs, forskolin, and PDE inhibitors, including selective inhibitors of PDE3, PDE4, PDE7, and PDE8, inhibit aggressive triple negative MDA-MB-231 breast cancer cell migration. Under the same conditions, these agents had little effect on breast cancer cell proliferation. This study demonstrates that PDE inhibitors inhibit breast cancer cell migration, and thus may be valuable therapeutic targets for inhibition of breast cancer metastasis. Since PDE8A is expressed in all breast cancer samples, and since dipyridamole, which inhibits PDE8, and PF-04957325, a selective PDE8 inhibitor, both inhibit migration, it suggests that PDE8A may be a valuable novel target for treatment of this disease.

Oral zinc aspartate treats experimental autoimmune encephalomyelitis.

The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.

Regulatory T-cells and cAMP suppress effector T-cells independently of PKA-CREM/ICER: a potential role for Epac.

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

Peroxisome proliferator-activated receptor gamma is required for CD4+ T cell-mediated lymphopenia-associated autoimmunity.

The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) was shown to play an immunoregulatory role in many immune-related cell types, and activation of PPARγ was reported to be an effective therapeutic approach in murine and human autoimmune disease. However, despite an association between lymphopenia and autoimmunity, there has been no study on the role of T cell PPARγ in lymphopenia-associated autoimmunity. In the present studies, we examined the role of PPARγ in CD4(+) T cells in two murine models of lymphopenia-associated autoimmunity. Surprisingly, we found that PPARγ expression in CD4(+) CD25(-) T cells (T effector cells [Teffs]) is actually required for development of autoimmunity under lymphopenic conditions. Mechanistically, the inability of PPARγ-deficient (T-PPAR) Teffs to mediate lymphopenic autoimmunity is associated with a significant decrease in accumulation of Teffs in the spleen, lymph nodes, and tissues after adoptive transfer. This abnormal accumulation of T-PPAR Teffs was associated with defects in both in vivo proliferation and survival. Additionally, T-PPAR Teffs demonstrated decreased cytokine production in inflammatory sites and decreased expression of the homing receptor α4ß7. Finally, these abnormalities in T-PPAR Teff function were not elicited by lymphopenia alone but also required the additional activation involved in the mediation of autoimmunity. Thus, in contrast to its documented immunosuppressive role, we identified an unexpected function for PPARγ in Teffs: a role in Teff proliferation and survival in lymphopenia-associated autoimmunity. These findings highlight both the multifunctional role of PPARγ in T cells and the complexity of PPARγ as a potential therapeutic target in autoimmunity.

Antigen-specific splenic CD4+ and CD8+ regulatory T cells generated via the eye, suppress experimental autoimmune encephalomyelitis either at the priming or at the effector phase.

The injection of antigen into the ocular anterior chamber (AC) induces the generation of splenic CD4(+) and CD8(+) regulatory T (Treg) cells, specific for the antigen injected into the AC. These Treg cells inhibit the induction (CD4(+)) and also the expression (CD8(+)) of a delayed-type hypersensitivity response. The ability of AC-induced self-antigen-specific Treg cells in modulating autoimmunity is not well defined. Here we show that an injection of encephalitogenic myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide into the anterior chamber of the eye (AC-MOG), before the induction of or during established experimental autoimmune encephalomyelitis (EAE) induced by MOG(35-55), suppresses the induction or progression of EAE, respectively. CD4(+) or CD8(+) splenic Treg cells induced by an injection of AC-MOG prevent EAE either at the inductive (priming) or at the progressive (effector) phase, respectively. This suppression of EAE by an AC-MOG injection or by intravenous transfer of splenic regulatory cells induced by an AC-MOG injection is specific for the antigen injected into the AC. Additionally, our data suggest that splenic CD8(+) Treg cells that suppress active EAE may use a transforming growth factor (TGF)-ß-dependent suppression mechanism while the suppression of the induction of EAE by the AC-induced CD4(+) Treg cells is independent of TGF-ß. Thus, we show for the first time that regulation of EAE at the priming or the chronic phase requires different phenotypes of Treg cells. Hence, it is important to consider the phenotype of Treg cells while designing effective cell-based therapies against autoimmune disorders.

Zinc aspartate suppresses T cell activation in vitro and relapsing experimental autoimmune encephalomyelitis in SJL/J mice.

Zinc is an essential trace element with a critical role in normal growth and development and in immune homeostasis. Zinc deficiency impairs both the innate and the adaptive immune system and can be normalized by zinc supplementation. On the other end of the spectrum, high dosages of zinc diminish immune cell functions similar to zinc deficiency. Here, we investigated the influence of zinc aspartate on proliferation and cytokine production of stimulated human T cells and mouse splenocytes in vitro. Furthermore, the effect of zinc aspartate was examined in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS) with a Th1/Th17 T cell-mediated immunopathogenesis. Zinc aspartate suppressed proliferation as well as IL-2, IL-10 and IL-17 production in stimulated human T cells and mouse splenocytes. Importantly, administration of a medium range dose of 30 µg/day zinc aspartate [1.5 mg/kg body weight (BW)] in a therapeutic manner led to a significant reduction of the clinical severity of the EAE during the first relapse of the disease. A lower zinc aspartate dose (6 µg/day, 0.3 mg/kg BW) had no significant therapeutic effect on the severity of the EAE, while administration of higher zinc aspartate amounts (120 µg/day, 6 mg/kg BW) led to more severe disease. Taken together, our data suggest that zinc aspartate can modulate activation, proliferation and cytokine production of effector T cells in vitro and in vivo and that activated autoreactive T cells may be potential therapeutic targets of tightly controlled zinc supplementation in autoimmune diseases like MS.

Treatment of Experimental Autoimmune Encephalomyelitis with an Inhibitor of Phosphodiesterase-8 (PDE8).

After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load.

PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice.

Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, 'peptidase-targeted Immunoregulation' (PETIR™), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR™ in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR™ represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.

The Role of PDE8 in T Cell Recruitment and Function in Inflammation.

Inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice to treat inflammatory disorders, with three PDE4 inhibitors currently in clinical use as therapeutics for psoriasis, psoriatic arthritis, atopic dermatitis and chronic obstructive pulmonary disease. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. It was shown that PDE8A plays a major role in controlling T cell and breast cancer cell motility, including adhesion to endothelial cells under physiological shear stress and chemotaxis. This is a unique function of PDE8 not shared by PDE4, another cAMP specific PDE, employed, as noted, as an anti-inflammatory therapeutic. Additionally, a regulatory role was shown for the PDE8A-rapidly accelerated fibrosarcoma (Raf)-1 kinase signaling complex in myelin antigen reactive CD4+ effector T cell adhesion and locomotion by a mechanism differing from that of PDE4. The PDE8A-Raf-1 kinase signaling complex affects T cell motility, at least in part, via regulating the LFA-1 integrin mediated adhesion to ICAM-1. The findings that PDE8A and its isoforms are expressed at higher levels in naive and myelin oligodendrocyte glycoprotein (MOG)35 - 55 activated effector T (Teff) cells compared to regulatory T (Treg) cells and that PDE8 inhibition specifically affects MOG35 - 55 activated Teff cell adhesion, indicates that PDE8A could represent a new beneficial target expressed in pathogenic Teff cells in CNS inflammation. The implications of this work for targeting PDE8 in inflammation will be discussed in this review.

Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin.

Statins, known as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, exhibit numerous functions related to inflammation, such as MHC class II down-regulation, interference with T cell adhesion, and induction of apoptosis. Here we demonstrate that both subcutaneous and oral administration of atorvastatin inhibit the development of actively induced chronic experimental autoimmune encephalomyelitis in SJL/J mice and significantly reduce the inflammatory infiltration into the central nervous system (CNS). When treatment was started after disease onset, atorvastatin reduced the incidence of relapses and protected from the development of further disability. Both the reduced autoreactive T cell response measured by proliferation toward the encephalitogenic peptide PLP139-151 and the cytokine profile indicate a potent blockade of T helper cell type 1 immune response. In in vitro assays atorvastatin not only inhibited antigen-specific responses, but also decreased T cell proliferation mediated by direct TCR engagement independently of MHC class II and LFA-1. Inhibition of proliferation was not due to apoptosis induction, but linked to a negative regulation on cell cycle progression. However, early T cell activation was unaffected, as reflected by unaltered calcium fluxes. Thus, our results provide evidence for a beneficial role of statins in the treatment of autoimmune attack on the CNS.

Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL.

Here, we provide evidence for a detrimental role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in neural death in T cell-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Clinical severity and neuronal apoptosis in brainstem motor areas were substantially reduced upon brain-specific blockade of TRAIL after induction of EAE through adoptive transfer of encephalitogenic T cells. Furthermore, TRAIL-deficient myelin-specific lymphocytes showed reduced encephalitogenicity when transferred to wild-type mice. Conversely, intracerebral delivery of TRAIL to animals with EAE increased clinical deficits, while naive mice were not susceptible to TRAIL. Using organotypic slice cultures as a model for living brain tissue, we found that neurons were susceptible to TRAIL-mediated injury induced by encephalitogenic T cells. Thus, in addition to its known immunoregulatory effects, the death ligand TRAIL contributes to neural damage in the inflamed brain.

Immunity to Respiratory Infection Is Reinforced Through Early Proliferation of Lymphoid TRM Cells and Prompt Arrival of Effector CD8 T Cells in the Lungs.

Cross-protection between serologically distinct strains of influenza A virus (IAV) is mediated by memory CD8 T cells that recognize epitopes from conserved viral proteins. Early viral control begins with activation of tissue-resident memory CD8 T cells (TRM) cells at the site of viral replication. These CD8 T cells do not act in isolation, as protection against disseminated infection is reinforced by multiple waves of effector cells (TEFF) that enter the lungs with different kinetics. To define how a protective CTL response evolves, we compared the functional properties of antiviral CD8 T cells in the respiratory tract and local lymphoid tissues. When analyzed 30 dpi, large numbers of antiviral CD8 T cells in the lungs and mediastinal lymph nodes (MLNs) expressed canonical markers of TRM cells (CD69 and/or CD103). The check point inhibitor PD-1 was also highly expressed on NP-specific CD8 T cells in the lungs, while the ratios of CD8 T cells expressing CD69 and CD103 varied according to antigen specificity. We next used in vitro experiments to identify conditions that induce a canonical TRM phenotype and found that that naïve and newly activated CD8 T cells maintain CD103 expression during culture with transforming growth factor-beta (TGFß), while central memory CD8 T cells (TCM) do not express CD103 under similar conditions. In vivo experiments showed that the distribution of antiviral CTLs in the MLN changed when immune mice were treated with reagents that block interactions with PD-L1. Importantly, the lymphoid TRM cells were poised for early proliferation upon reinfection with a different strain of IAV and defenses in the lungs were augmented by a transient increase in numbers of TEFF cells at the site of infection. As the interval between infections increased, lymphoid TRM cells were replaced with TCM cells which proliferated with delayed kinetics and contributed to an exaggerated inflammatory response in the lungs.

Prophylactic and therapeutic suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor.

The objective was to optimize and evaluate the in vivo activities of our novel bifunctional peptide inhibitor (BPI), which alters immune response in autoimmune diseases by modulating the immunological synapse formation. Previously, we have designed PLP-BPI and GAD-BPI by conjugating myelin proteolipid protein (PLP)(139-151) and glutamic acid decarboxylase (GAD)(208-217), respectively, with CD11a(237-246) via a spacer peptide. PLP-BPI and GAD-BPI suppressed the disease progression in experimental autoimmune encephalomyelitis (EAE) and in type-1 diabetes, respectively. In this study, various PLP-BPI derivatives were synthesized and evaluated in the EAE model. Intravenous injections of PLP-BPI derivatives prevented the disease progression more efficiently than did unmodified PLP-BPI. Production of IL-17, a potent proinflammatory cytokine found commonly among MS patients, was significantly low in Ac-PLP-BPI-NH(2)-2-treated mice. Treatment given after the disease onset could dramatically ameliorate the disease. BPI induced anaphylactic responses at a lower incidence than PLP(139-151). In conclusion, PLP-BPI derivatives can effectively suppress the disease severity and morbidity of EAE by post-onset therapeutic treatment as well as prophylactic use.

DP IV/CD26, APN/CD13 and related enzymes as regulators of T cell immunity: implications for experimental encephalomyelitis and multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system. Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) play a regulatory role in T cell activation and represent potential targets for the treatment of inflammatory disorders. Synthetic inhibitors of DP IV and/or APN enzymatic activity induce production of the immunosuppressive cytokine TGF-beta1 and subsequently suppress DNA synthesis and Th1 cytokine production of activated human T cells. Compelling evidence has demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to the pathogenesis of autoimmune inflammation. Here, we report that inhibitors of DP IV-like activity as well as of APN activity inhibit IL-17 production in activated human and mouse T cells. Combining inhibitors of DP IV and APN increases the suppressive effect on T cell specific IL-17 production in vitro compared to a single peptidase inhibitor. In the following, we summarize the evidence for the role of both ectoenzymes in T cell activation in vitro and in vivo and provide a rationale for the use of combined or dual ectopeptidase inhibitors to treat autoimmune diseases like MS.

Dual inhibition of dipeptidyl peptidase IV and aminopeptidase N suppresses inflammatory immune responses.

The ectopeptidases dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) are known to regulate T cell activation. Since selective inhibitors of DP IV and APN suppress DNA synthesis and cytokine production of stimulated T cells in a TGF-beta1-dependent manner, we tested whether combined application of DP IV and APN inhibitors enhances this immunomodulatory effect. The results show that simultaneous application of DP IV and APN inhibitors significantly suppressed DNA synthesis in mitogen- or anti-CD3-stimulated human T cells in vitro when compared to the use of a single DP IV or APN inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting both DP IV and APN led to a potent treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). This review summarizes the evidence for the role of both enzymes in T cell activation in vitro and in vivo and provides a rationale for using combined and dual peptidase inhibitors to treat autoimmune diseases like MS.

PDE8 controls CD4+ T cell motility through the PDE8A-Raf-1 kinase signaling complex.

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4+ T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells, we used a cell permeable peptide that selectively disrupts the PDE8A-Raf-1 interaction. The disruptor peptide inhibits the Teff-endothelial cell interaction more potently than the enzymatic inhibitor. Furthermore, the LFA-1/ICAM-1 interaction was identified as a target of disruptor peptide mediated reduction of adhesion, spreading and locomotion of Teff cells under flow. Mechanistically, we observed that disruption of the PDE8A-Raf-1 complex profoundly alters Raf-1 signaling in Teff cells. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase complex disruptors decreases Teff cell adhesion and migration under flow, and represents a novel approach to target T cells in inflammation.

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation.

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.

Differential Expression and Function of PDE8 and PDE4 in Effector T cells: Implications for PDE8 as a Drug Target in Inflammation.

Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40-100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4(+) Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4(+) Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4(+) T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a potential drug target, we compared the selective and combined effects of the recently characterized highly potent PDE8-selective inhibitor PF-04957325 with the PDE4-selective inhibitor piclamilast (PICL). As previously shown, PF-04957325 suppresses T cell adhesion to endothelial cells. In contrast, we found that PICL alone increased firm T cell adhesion to endothelial cells by ~20% and significantly abrogated the inhibitory effect of PF-04957325 on T cell adhesion by over 50% when cells were co-exposed to PICL and PF-04957325. Despite its robust effect on T cell adhesion, PF-04957325 was over two orders of magnitude less efficient than PICL in suppressing polyclonal Teff cell proliferation, and showed no effect on cytokine gene expression in these cells. More importantly, PDE8 inhibition did not suppress proliferation and cytokine production of myelin-antigen reactive proinflammatory Teff cells in vivo and in vitro. Thus, targeting PDE8 through PF-04957325 selectively regulates Teff cell interactions with endothelial cells without marked immunosuppression of proliferation, while PDE4 inhibition has partially opposing effects. Collectively, our data identify PF-04957325 as a novel function-specific tool for the suppression of Teff cell adhesion and indicate that PDE4 and PDE8 play unique and non-redundant roles in the control of Teff cell functions.