RESUMEN
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.
Asunto(s)
Modulador del Elemento de Respuesta al AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , AMP Cíclico/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Tolerancia Inmunológica/fisiología , Linfocitos T Reguladores/inmunología , Animales , Proliferación Celular/fisiología , AMP Cíclico/genética , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Ratones Noqueados , Linfocitos T Reguladores/citologíaRESUMEN
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.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfopenia/inmunología , PPAR gamma/inmunología , Traslado Adoptivo , Animales , Apoptosis/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Separación Celular , Citocinas/biosíntesis , Femenino , Citometría de Flujo , Linfopenia/metabolismo , Ratones , Ratones Noqueados , PPAR gamma/metabolismoRESUMEN
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.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Animales , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/metabolismo , Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Células Th17RESUMEN
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.
RESUMEN
BACKGROUND: Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. METHODOLOGY/PRINCIPAL FINDINGS: We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem-/- mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. CONCLUSION/SIGNIFICANCE: Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.