Early leukocyte activation receptor CD69: a novel player in the maintenance of the Th17/Treg balance in peritoneal fibrosis

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 09-03-2015CD69 is induced after activation of leukocytes at inflammatory sites. Mice lacking CD69 develop exacerbated forms of inflammatory diseases, which are largely mediated by misbalanced responses of T helper (Th) 17 cells and regulatory T cells (Tregs), indicating that CD69 modulates Th17 differentiation and plays a role in regulatory T cell function. However, the pathophysiological role of CD69 in fibrotic diseases remains largely unknown. Renal patients subjected to peritoneal dialysis (PD) develop progressive peritoneal fibrosis, which may lead to technique failure. Herein, we observed that infiltrating T lymphocytes of PD patients expressed high levels of CD69. Thus, we explored the role of CD69 in fibro-proliferative responses by analyzing a model of peritoneal fibrosis induced by dialysis fluid exposure in WT and CD69-deficient mice. CD69–/– mice showed enhanced peritoneal thickness and myofibroblasts accumulation as well as higher incidence of mesothelial to mesenchymal transition (MMT). In parallel, CD69–/– mice showed exacerbated inflammatory infiltrates, a marked increase of Th17 cells and IL-17 cytokine and reduction of Tregs. Transplantation of a mixture of bone marrows from CD69–/– and Rag2–/– mice into WT recipients reproduced the severity of the disease upon PD fluid exposure, demonstrating that CD69 exerts its function within the lymphocyte compartment. Blockade of IL-17 in CD69–/– mice reduced Th17 response and inflammatory infiltrates and resulted in decreased peritoneal fibrosis induced by PD fluid. Conversely, blockade of CD69 in WT mice mimicked the exacerbated response of CD69–/– mice to PD fluid exposure. Our findings indicate that CD69 modulates Th17-mediated inflammatory responses in the peritoneal cavity and negatively regulates peritoneal fibrosis induced by dialysis fluid exposure.El receptor CD69 se induce tras la activación leucocitaria en los infiltrados inflamatorios. Los ratones deficientes en CD69 desarrollan formas exacerbadas de enfermedades inflamatorias que están mediadas por una respuesta desbalanceada entre linfocitos T “helper” Th17 y células T reguladora (Tregs), lo que indica que CD69 modula la diferenciación de Th17 y que juega un papel en la función de las células Tregs. Sin embargo, el papel fisio-patológico de CD69 en enfermedades fibróticas es desconocido. Los pacientes renales sometidos a diálisis peritoneal (DP) desarrollan fibrosis peritoneal progresiva, la cual puede dar lugar al fallo de la técnica. En este estudio, hemos observado altos niveles de expresión de CD69 en los linfocitos infiltrantes de los pacientes en DP. Por lo tanto, decidimos explorar el papel de CD69 en la respuesta fibroproliferativa mediante el análisis de un modelo de fibrosis peritoneal inducido por líquidos de diálisis en ratones silvestres y en ratones deficientes para CD69. Los ratones CD69–/– mostraron un mayor engrosamiento peritoneal y una mayor acumulación de miofibroblastos, así como una mayor incidencia de transición mesotelio mesenquimal (TMM). En paralelo, los ratones CD69–/– mostraron infiltrados inflamatorios exacerbados, un marcado aumento de células Th17 y de la citoquina IL-17 y una reducción de células Tregs. El trasplante de una mezcla de medulas óseas procedentes de ratones CD69–/– y Rag2–/– en ratones silvestres reprodujo la severidad de la enfermedad inducida por la exposición al líquido de DP, demostrando asi que CD69 ejerce su función en el compartimento de linfocitos. El bloqueo de IL- 17 en ratones CD69–/– redujo la respuesta Th17 y el infiltrado inflamatorio y dio lugar a una reducción de la fibrosis inducida por el líquido de DP. Por otro lado, el bloqueo de CD69 en ratones silvestres mimetizó la respuesta exacerbada a la exposición del liquido de diálisis de los ratones CD69–/–. Nuestros resultados indican que CD69 modula la respuesta inflamatoria mediada por Th17 en la cavidad peritoneal y regula negativamente la fibrosis peritoneal inducida por la exposición a liquido de diálisi

A Novel Mouse Model of Peritoneal Dialysis: Combination of Uraemia and Long-Term Exposure to PD Fluid

Different animal models for peritoneal dialysis (PD) have been used in the past decades to develop PD fluids compatible with patient life and to identify markers of peritoneal fibrosis and inflammation. Only few of those studies have taken into account the importance of uraemia-induced alterations at both systemic and peritoneal levels. Moreover, some animal studies which have reported about PD in a uremic setting did not always entirely succeed in terms of uraemia establishment and animal survival. In the present study we induced uraemia in the recently established mouse PD exposure model in order to obtain a more clinically relevant mouse model for kidney patients. This new designed model reflected both the slight thickening of peritoneal membrane induced by uraemia and the significant extracellular matrix deposition due to daily PD fluid instillation. In addition the model offers the opportunity to perform long-term exposure to PD fluids, as it is observed in the clinical setting, and gives the advantage to knock out candidate markers for driving peritoneal inflammatory mechanisms.Marie Curie actionsPeer Reviewe

Paricalcitol reduces peritoneal fibrosis in mice through the activation of regulatory T cells and reduction in IL-17 production

Fibrosis is a significant health problem associated with a chronic inflammatory reaction. The precise mechanisms involved in the fibrotic process are still poorly understood. However, given that inflammation is a major causative factor, immunomodulation is a possible therapeutic approach to reduce fibrosis. The vitamin D receptor (VDR) that is present in all hematopoietic cells has been associated with immunomodulation. We investigated whether the intraperitoneal administration of paricalcitol, a specific activator of the VDR, modulates peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis. We characterized the inflammatory process in the peritoneal cavity of mice treated or not treated with paricalcitol and analyzed the ensuing fibrosis. The treatment reduced peritoneal IL-17 levels, which strongly correlated with a significantly lower peritoneal fibrotic response. In vitro studies demonstrate that both CD4+ and CD8+ regulatory T cells appear to impact the regulation of IL-17. Paricalcitol treatment resulted in a significantly increased frequency of CD8+ T cells showing a regulatory phenotype. The frequency of CD4+ Tregs tends to be increased, but it did not achieve statistical significance. However, paricalcitol treatment increased the number of CD4+ and CD8+ Treg cells in vivo. In conclusion, the activation of immunological regulatory mechanisms by VDR signaling could prevent or reduce fibrosis, as shown in peritoneal fibrosis induced by PDF exposure in mice.This study was supported by RETICS 06/0016 (VFM, RS) and FIS PI 09/0064 (RS) from the Fondo de Investigaciones Sanitarias (Health Research Fund). MLC was funded by SAF 2013-47611-R, SAF 2010-21249, and SAF 2007-61201 from the Ministerio de Economía y competitividad. MRO was supported by RETICS 12/0021,S2012DMD2321 from the Comunidad Autónoma de Madrid, PI 11/01854 from Fondo Investigaciones Sanitarias. GTGM was supported by Renal Foundation Íñigo Álvarez de Toledo, FIBHULP, and by Severo Ochoa FoundationPeer Reviewe

Rapamycin Protects from Type-I Peritoneal Membrane Failure Inhibiting the Angiogenesis, Lymphangiogenesis, and Endo-MT

Preservation of peritoneal membrane (PM) is essential for long-term survival in peritoneal dialysis (PD). Continuous presence of PD fluids (PDF) in the peritoneal cavity generates chronic inflammation and promotes changes of the PM, such as fibrosis, angiogenesis, and lymphangiogenesis. Mesothelial-to-mesenchymal transition (MMT) and endothelial-to-mesenchymal transition (Endo-MT) seem to play a central role in this pathogenesis. We speculated that Rapamycin, a potent immunosuppressor, could be beneficial by regulating blood and lymphatic vessels proliferation. We demonstrate that mice undergoing a combined PD and Rapamycin treatment (PDF + Rapa group) presented a reduced PM thickness and lower number of submesothelial blood and lymphatic vessels, as well as decreased MMT and Endo-MT, comparing with their counterparts exposed to PD alone (PDF group). Peritoneal water transport in the PDF + Rapa group remained at control level, whereas PD effluent levels of VEGF, TGF-β, and TNF-α were lower than in the PDF group. Moreover, the treatment of mesothelial cells with Rapamycin in vitro significantly decreased VEGF synthesis and selectively inhibited the VEGF-C and VEGF-D release when compared with control cells. Thus, Rapamycin has a protective effect on PM in PD through an antifibrotic and antiproliferative effect on blood and lymphatic vessels. Moreover, it inhibits Endo-MT and, at least partially, MMT.“Fondo de Investigaciones Sanitarias” (FIS), Instituto Carlos-III to Abelardo Aguilera, SAF2013-47611R from the “Ministerio de Economia y Competitividad”, and S2010/BMD-2321 (FIBROTEAM Consortium) from “Comunidad Autónoma de Madrid” to Manuel López-CabreraPeer Reviewe

Paricalcitol reduces peritoneal fibrosis in mice through the activation of regulatory T cells and reduction in IL-17 production.

Fibrosis is a significant health problem associated with a chronic inflammatory reaction. The precise mechanisms involved in the fibrotic process are still poorly understood. However, given that inflammation is a major causative factor, immunomodulation is a possible therapeutic approach to reduce fibrosis. The vitamin D receptor (VDR) that is present in all hematopoietic cells has been associated with immunomodulation. We investigated whether the intraperitoneal administration of paricalcitol, a specific activator of the VDR, modulates peritoneal dialysis fluid (PDF)-induced peritoneal fibrosis. We characterized the inflammatory process in the peritoneal cavity of mice treated or not treated with paricalcitol and analyzed the ensuing fibrosis. The treatment reduced peritoneal IL-17 levels, which strongly correlated with a significantly lower peritoneal fibrotic response. In vitro studies demonstrate that both CD4+ and CD8+ regulatory T cells appear to impact the regulation of IL-17. Paricalcitol treatment resulted in a significantly increased frequency of CD8+ T cells showing a regulatory phenotype. The frequency of CD4+ Tregs tends to be increased, but it did not achieve statistical significance. However, paricalcitol treatment increased the number of CD4+ and CD8+ Treg cells in vivo. In conclusion, the activation of immunological regulatory mechanisms by VDR signaling could prevent or reduce fibrosis, as shown in peritoneal fibrosis induced by PDF exposure in mice

T Helper 17/Regulatory T Cell Balance and Experimental Models of Peritoneal Dialysis-Induced Damage

Fibrosis is a general complication in many diseases. It is the main complication during peritoneal dialysis (PD) treatment, a therapy for renal failure disease. Local inflammation and mesothelial to mesenchymal transition (MMT) are well known key phenomena in peritoneal damage during PD. New data suggest that, in the peritoneal cavity, inflammatory changes may be regulated at least in part by a delicate balance between T helper 17 and regulatory T cells. This paper briefly reviews the implication of the Th17/Treg-axis in fibrotic diseases. Moreover, it compares current evidences described in PD animal experimental models, indicating a loss of Th17/Treg balance (Th17 predominance) leading to peritoneal damage during PD. In addition, considering the new clinical and animal experimental data, new therapeutic strategies to reduce the Th17 response and increase the regulatory T response are proposed. Thus, future goals should be to develop new clinical biomarkers to reverse this immune misbalance and reduce peritoneal fibrosis in PD.This work was supported in part by grants from Ministerio de Economia y competitividad SAF2010-21249 to Manuel López-Cabrera, Comunidad Autónoma de Madrid 2010-BMD2321 (FIBROTEAM) to Manuel Lopez Cabrera, and Fondo de Investigaciones Santitarias RETICS 06/0016 and PI 09/0064 to Rafael Selgas and FIS 12/01175 to Abelardo Aguilera Peralta. Georgios Liappas is fully supported from European Union, Seventh Framework Program “EuTRiPD,” under Grant Agreement PITN-GA-2011-287813. The authors would like to thank Juliette Siegfried and her team at ServingMed.com for editing the language of the paper.Peer Reviewe

Paricalcitol reduced peritoneal membrane fibrosis, inflammation and ultrafiltration failure in mice exposed to PDF.

<p>A) Paraffin sections of the peritoneal membrane from the 3 groups were stained with Masson's trichrome. B) Thickening of the peritoneal membrane was determined by morphometric analysis. C) Peritoneal permeability was determined by net ultrafiltration. D) The presence of inflammatory and mesothelial cells was determined by the expression of CD45 (green) and cytokeratin (red), respectively, in frozen sections of peritoneal membrane representative of each group. A green arrow indicates hematopoietic cells. A red arrow indicates mesothelial cells. E) The angiogenesis was determined by the expression of CD31 (green). Cytokeratin-positive cells are stained in red. The color balance was equally adjusted in immunofluorescence using Photoshop V10 for Mac (Abobe Systems Incorporated, US). n≥5 in each group. Statistical significance was determined using the Mann-Whitney test. *<i>P</i><.05; ***<i>P</i><.001.</p

CD4+ and CD8+ T cells from paricalcitol-treated mice regulate IL-17 production.

<p>CD4⁺- and CD8⁺-enriched T cell populations from mice treated with PDF and paricalcitol were stained with PKH67 and co-cultured with CD3⁺ T cells from PDF-treated mice. A control group of CD3⁺ T enriched cells from PDF-treated mice was also included. The cells were stimulated with CD3 and CD28, and after 2 days, brefeldin A was added for the last 12 h of culture. The cells were stained with anti-CD4 and anti-IL-17 antibodies and analyzed by flow cytometry. The PKH67^negative CD4⁺ T cells were selected, and the frequency of IL17⁺ cells is shown in the figure. Statistical significance was determined using the Mann-Whitney test. **<i>P</i><.01.</p