Browsing by Author "Prado, Carolina"

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    Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity
    (2021) Prado, Carolina; Osorio Barrios, Francisco; Falcón, Paulina; Espinoza, Alexandra; Saez, Juan José; Yuseff Sepúlveda, María Isabel; Pacheco, Rodrigo
    Abstract Background Recent evidence has shown dopamine as a major regulator of inflammation. Accordingly, dopaminergic regulation of immune cells plays an important role in the physiopathology of inflammatory disorders. Multiple sclerosis (MS) is an inflammatory disease involving a CD4+ T-cell-driven autoimmune response to central nervous system (CNS) derived antigens. Evidence from animal models has suggested that B cells play a fundamental role as antigen-presenting cells (APC) re-stimulating CD4+ T cells in the CNS as well as regulating T-cell response by mean of inflammatory or anti-inflammatory cytokines. Here, we addressed the role of the dopamine receptor D3 (DRD3), which displays the highest affinity for dopamine, in B cells in animal models of MS. Methods Mice harbouring Drd3-deficient or Drd3-sufficient B cells were generated by bone marrow transplantation into recipient mice devoid of B cells. In these mice, we compared the development of experimental autoimmune encephalomyelitis (EAE) induced by immunization with a myelin oligodendrocyte glycoprotein (MOG)-derived peptide (pMOG), a model that leads to CNS-autoimmunity irrespective of the APC-function of B cells, or by immunization with full-length human MOG protein (huMOG), a model in which antigen-specific activated B cells display a fundamental APC-function in the CNS. APC-function was assessed in vitro by pulsing B cells with huMOG-coated beads and then co-culturing with MOG-specific T cells. Results Our data show that the selective Drd3 deficiency in B cells abolishes the disease development in the huMOG-induced EAE model. Mechanistic analysis indicates that although DRD3-signalling did not affect the APC-function of B cells, DRD3 favours the CNS-tropism in a subset of pro-inflammatory B cells in the huMOG-induced EAE model, an effect that was associated with higher CXCR3 expression. Conversely, the results show that the selective Drd3 deficiency in B cells exacerbates the disease severity in the pMOG-induced EAE model. Further analysis shows that DRD3-stimulation increased the expression of the CNS-homing molecule CD49d in a B-cell subset with anti-inflammatory features, thus attenuating EAE manifestation in the pMOG-induced EAE model. Conclusions Our findings demonstrate that DRD3 in B cells exerts a dual role in CNS-autoimmunity, favouring CNS-tropism of pro-inflammatory B cells with APC-function and promoting CNS-homing of B cells with anti-inflammatory features. Thus, these results show DRD3-signalling in B cells as a critical regulator of CNS-autoimmunity.
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    Endogenous Galectin-8 protects against Th17 infiltration and fibrosis following acute kidney injury
    (Springer Nature, 2025) Perez-Moreno, Elisa; Peña, Adely de la; Toledo, Tomás; Saez, Javiera; Pérez-Molina, Francisca; Espinoza, Sofía; Metz, Claudia; Díaz-Valdivia, Nicole; Azócar, Lorena; Prado, Carolina; Pacheco, Rodrigo; Segovia-Miranda, Fabian; Godoy, Alejandro S.; Amador, Cristian A.; Feuerhake González, Teo; González, Alfonso; Soza, Andrea
    Background Acute kidney injury (AKI) is a serious clinical condition characterized by a rapid decline in renal function, often progressing to chronic kidney disease (CKD) and fibrosis. The endogenous mechanisms influencing kidney injury resolution or maladaptive repair remain poorly understood. Galectin‑8 (Gal‑8), a tandem‑repeat β‑galactosidebinding lectin, plays a role in epithelial cell proliferation, epithelial‑mesenchymal transition, and immune regulation, all of which are critical in AKI outcomes. While exogenous Gal‑8 administration has shown renoprotective effects, its endogenous role in kidney injury progression and resolution remains unclear. Methods To investigate the endogenous role of Gal‑8 in AKI, we compared the responses of Gal‑8 knockout (Gal8‑KO; Lgals8−/− bearing a β‑gal cassette under the Lgals8 gene promoter) and wild‑type (Lgals8+/+) mice in a nephrotoxic folic acid (FA)‑induced AKI model. Renal Gal‑8 expression was assessed by β‑galactosidase staining, lectin‑marker colocalization, and RT‑qPCR. Renal function, structure, and immune responses were evaluated at the acute (day 2) and fibrotic (day 14) phases of injury. Plasma creatinine levels were measured to assess renal function, while histological analyses evaluated tubular damage, renal inflammation, and extracellular matrix deposition. Flow cytometry was performed to characterize the immune response, focusing on pro‑inflammatory T cells. Results Galectin‑8 was predominantly expressed in the renal cortex, localizing to tubules, glomeruli, and blood vessels, with its levels decreasing by half following AKI. Both Lgals8+/+ and Lgals8−/− mice exhibited similar renal function and structure impairments during the acute phase, though Lgals8+/+ mice showed slightly worse damage. By the fibrotic phase, Lgals8−/− mice exhibited more pronounced cortical damage and fibrosis, characterized by increased type I and III collagen deposition and enhanced Th17 cell infiltration, while myofibroblast activation remained comparable to that of Lgals8+/+ mice. Conclusions Endogenous Gal‑8 does not significantly protect the kidney during the acute phase and is dispensable for cell proliferation and death in response to AKI. However, it is crucial in preventing maladaptive repair by regulating.
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    GPR43 stimulation on TCRαβ+ intraepithelial colonic lymphocytes inhibits the recruitment of encephalitogenic T-cells into the central nervous system and attenuates the development of autoimmunity
    (2023) Prado, Carolina; Espinoza, Alexandra; Martínez-Hernández, J. E.; Petrosino, Joseph; Riquelme Sánchez, Erick Marcelo; Martin, Alberto J. M.; Pacheco, Rodrigo
    Abstract Introduction Gut microbiota plays a critical role in the regulation of immune homeostasis. Accordingly, several autoimmune disorders have been associated with dysbiosis in the gut microbiota. Notably, the dysbiosis associated with central nervous system (CNS) autoimmunity involves a substantial reduction of bacteria belonging to Clostridia clusters IV and XIVa, which constitute major producers of short-chain fatty acids (SCFAs). Here we addressed the role of the surface receptor-mediated effects of SCFAs on mucosal T-cells in the development of CNS autoimmunity. Methods To induce CNS autoimmunity, we used the mouse model of experimental autoimmune encephalomyelitis (EAE) induced by immunization with the myelin oligodendrocyte glycoprotein (MOG)-derived peptide (MOG35-55 peptide). To address the effects of GPR43 stimulation on colonic TCRαβ+ T-cells upon CNS autoimmunity, mucosal lymphocytes were isolated and stimulated with a selective GPR43 agonist ex vivo and then transferred into congenic mice undergoing EAE. Several subsets of lymphocytes infiltrating the CNS or those present in the gut epithelium and gut lamina propria were analysed by flow cytometry. In vitro migration assays were conducted with mucosal T-cells using transwells. Results Our results show a sharp and selective reduction of intestinal propionate at the peak of EAE development, accompanied by increased IFN-γ and decreased IL-22 in the colonic mucosa. Further analyses indicated that GPR43 was the primary SCFAs receptor expressed on T-cells, which was downregulated on colonic TCRαβ+ T-cells upon CNS autoimmunity. The pharmacologic stimulation of GPR43 increased the anti-inflammatory function and reduced the pro-inflammatory features in several TCRαβ+ T-cell subsets in the colonic mucosa upon EAE development. Furthermore, GPR43 stimulation induced the arrest of CNS-autoreactive T-cells in the colonic lamina propria, thus avoiding their infiltration into the CNS and dampening the disease development. Mechanistic analyses revealed that GPR43-stimulation on mucosal TCRαβ+ T-cells inhibits their CXCR3-mediated migration towards CXCL11, which is released from the CNS upon neuroinflammation. Conclusions These findings provide a novel mechanism involved in the gut-brain axis by which bacterial-derived products secreted in the gut mucosa might control the CNS tropism of autoreactive T-cells. Moreover, this study shows GPR43 expressed on T-cells as a promising therapeutic target for CNS autoimmunity.
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    GPR43 stimulation on TCRαβ+ intraepithelial colonic lymphocytes inhibits the recruitment of encephalitogenic T-cells into the central nervous system and attenuates the development of autoimmunity
    (2023) Prado, Carolina; Espinoza, Alexandra; Martinez-Hernandez, J. Eduardo; Petrosino, Joseph; Riquelme, Erick; Martin, Alberto J. M.; Pacheco, Rodrigo
    IntroductionGut microbiota plays a critical role in the regulation of immune homeostasis. Accordingly, several autoimmune disorders have been associated with dysbiosis in the gut microbiota. Notably, the dysbiosis associated with central nervous system (CNS) autoimmunity involves a substantial reduction of bacteria belonging to Clostridia clusters IV and XIVa, which constitute major producers of short-chain fatty acids (SCFAs). Here we addressed the role of the surface receptor-mediated effects of SCFAs on mucosal T-cells in the development of CNS autoimmunity.MethodsTo induce CNS autoimmunity, we used the mouse model of experimental autoimmune encephalomyelitis (EAE) induced by immunization with the myelin oligodendrocyte glycoprotein (MOG)-derived peptide (MOG(35-55) peptide). To address the effects of GPR43 stimulation on colonic TCR alpha beta(+) T-cells upon CNS autoimmunity, mucosal lymphocytes were isolated and stimulated with a selective GPR43 agonist ex vivo and then transferred into congenic mice undergoing EAE. Several subsets of lymphocytes infiltrating the CNS or those present in the gut epithelium and gut lamina propria were analysed by flow cytometry. In vitro migration assays were conducted with mucosal T-cells using transwells.ResultsOur results show a sharp and selective reduction of intestinal propionate at the peak of EAE development, accompanied by increased IFN-gamma and decreased IL-22 in the colonic mucosa. Further analyses indicated that GPR43 was the primary SCFAs receptor expressed on T-cells, which was downregulated on colonic TCR alpha beta(+) T-cells upon CNS autoimmunity. The pharmacologic stimulation of GPR43 increased the anti-inflammatory function and reduced the pro-inflammatory features in several TCR alpha beta(+) T-cell subsets in the colonic mucosa upon EAE development. Furthermore, GPR43 stimulation induced the arrest of CNS-autoreactive T-cells in the colonic lamina propria, thus avoiding their infiltration into the CNS and dampening the disease development. Mechanistic analyses revealed that GPR43-stimulation on mucosal TCR alpha beta(+) T-cells inhibits their CXCR3-mediated migration towards CXCL11, which is released from the CNS upon neuroinflammation.ConclusionsThese findings provide a novel mechanism involved in the gut-brain axis by which bacterial-derived products secreted in the gut mucosa might control the CNS tropism of autoreactive T-cells. Moreover, this study shows GPR43 expressed on T-cells as a promising therapeutic target for CNS autoimmunity.
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    Identification of master regulator genes controlling pathogenic CD4+ T cell fate in inflammatory bowel disease through transcriptional network analysis
    (2024) Jimenez, Jose M.; Contreras-Riquelme, J. Sebastian; Vidal, Pia M.; Prado, Carolina; Bastias, Macarena; Meneses, Claudio; Martin, Alberto J. M.; Perez-Acle, Tomas; Pacheco, Rodrigo
    Inflammatory bowel diseases (IBD) are a group of chronic inflammatory conditions of the gastrointestinal tract associated with multiple pathogenic factors, including dysregulation of the immune response. Effector CD4(+) T cells and regulatory CD4(+) T cells (Treg) are central players in maintaining the balance between tolerance and inflammation. Interestingly, genetic modifications in these cells have been implicated in regulating the commitment of specific phenotypes and immune functions. However, the transcriptional program controlling the pathogenic behavior of T helper cells in IBD progression is still unknown. In this study, we aimed to find master transcription regulators controlling the pathogenic behavior of effector CD4(+) T cells upon gut inflammation. To achieve this goal, we used an animal model of IBD induced by the transfer of na & iuml;ve CD4(+) T cells into recombination-activating gene 1 (Rag1) deficient mice, which are devoid of lymphocytes. As a control, a group of Rag1 -/- mice received the transfer of the whole CD4(+) T cells population, which includes both effector T cells and Treg. When gut inflammation progressed, we isolated CD4(+) T cells from the colonic lamina propria and spleen tissue, and performed bulk RNA-seq. We identified differentially up- and down-regulated genes by comparing samples from both experimental groups. We found 532 differentially expressed genes (DEGs) in the colon and 30 DEGs in the spleen, mostly related to Th1 response, leukocyte migration, and response to cytokines in lamina propria T-cells. We integrated these data into Gene Regulatory Networks to identify Master Regulators, identifying four up-regulated master gene regulators (Lef1, Dnmt1, Mybl2, and Jup) and only one down-regulated master regulator (Foxo3). The altered expression of master regulators observed in the transcriptomic analysis was confirmed by qRT-PCR analysis and found an up-regulation of Lef1 and Mybl2, but without differences on Dnmt1, Jup, and Foxo3. These two master regulators have been involved in T cells function and cell cycle progression, respectively. We identified two master regulator genes associated with the pathogenic behavior of effector CD4(+) T cells in an animal model of IBD. These findings provide two new potential molecular targets for treating IBD.
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    Smoking promotes exacerbated inflammatory features in dendritic cells of Chilean rheumatoid arthritis patients
    (2018) Prado, Carolina; Iruretagoyena, Mirentxu; Burgos, Paula, I; Pacheco, Rodrigo
    Background: The dual potential to promote tolerance or inflammation when facing self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. There is an association between smoking and DCs maturation in patients with rheumatoid arthritis (RA). However, ethnicity is a key factor in autoimmune disorders. Aim: To evaluate phenotypic and functional alterations of DCs obtained from Chilean patients with RA as compared to healthy controls (HC). In second term, to compare the inflammatory behaviour of DCs between smoker and non-smoker patients. Material and Methods: Monocyte-derived DCs and T-cells were obtained from blood samples isolated from 30 HC and 32 RA-patients, 14 of which were currently smokers and 18 non-smokers. Several maturation surface markers were evaluated in DCs, including HLA-DR, CD40, CD80, CD83 and CD86. Furthermore, autologous co-cultures of DCs and T-cells were carried out and then T-cell proliferation, and expansion of Th1, Th17 and Tregs were analysed. Results: Compared with HC, RA-patients displayed increased HLA-DR expression in DCs, which was manifested mainly in patients with moderate-to- high disease activity scores (DAS28). Furthermore, RA-patients presented a stronger Th17-expansion and a correlation between DAS28 and Th1-expansion. Both effects were mainly observed in patients in remission or with a low DAS28. Moreover, smoker RA-patients displayed enhanced HLA-DR and CD83 expression in DCs as well as an exacerbated Th17-expansion and a correlation between DAS28 and Th1-expansion. Conclusions: These findings suggest that smoking enhances the inflammatory behaviour of DCs and the consequent Th1 and Th17-mediated response in patients with RA
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    Smoking promotes exacerbated inflammatory features in dendritic cells of Chilean rheumatoid arthritis patients
    (2018) Prado, Carolina; Iruretagoyena B., Mirentxu; Burgos, Paula I.; Pacheco, Rodrigo