Eugenio Contreras Castillo

Mexico

TIF1? regulates stability of regulatory T cells during inflammation

Eugenio Contreras-Castillo1., Jesús Daniel Zambrano-Romero1., Diego Pérez-Vázquez1., María Guadalupe García-Patiño1., Javier Orozco-Cordoba1., Ofelia Muñoz-Paleta1., Gustavo Tapia-Urzúa2., Diego Delgado-Zaldívar3., Ma. Cleofas Marcial-Medina1., Aurora Candelario-Martínez4., Jose Luis Ramos-Balderas1., Irma Ileana Licona-Limón5., Isaac Martínez-Racine6, Iris Madera-Salcedo3., Florencia Rosetti3., Jose C Crispin3,7., Lilia Noriega-López8., Porfirio Nava4., Araceli Pérez-López9., Félix Recillas-Targa2., Vesa Kaartinen10., Jorge Henao-Mejía11., Richard Flavell5,12., Paula Licona-Limón1*.

1 Departamento de Biología Celular y del Desarrollo, Instituto de Fisiología Celular. Universidad Nacional Autónoma de México. 04510, Mexico City.
2 Departamento de Genética Molecular, Instituto de Fisiología Celular. Universidad Nacional Autónoma de México. 04510, Mexico City.
3 Departamento de Inmunologia y Reumatologia, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. 14080, Mexico City. 4 Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV-IPN. 07360 Mexico City. 5 Department of Immunobiology, Yale University, New Haven, CT, 06520, USA. 6 Departamento de Patología, Facultad de Medicina Veterinaria y Zootecnia UNAM 7 Escuela de Medicina y Ciencias de la Salud, Tecnológico de Monterrey, 64849, Monterrey, Mexico 8 Fisiología de la Nutrición, Instituto Nacional de Ciencias Medicas y Nutrición Salvador Zubirán. 14080, Mexico City.
9 Unidad de Investigación en Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México. 54090, Mexico City.
10 Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, 48104, USA. 11 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Division of Protective Immunity, Department of Pathology and Laboratory Medicine, Children’s Hospital of Pennsylvania, University of Pennsylvania, Philadelphia, PA 19104, USA. 12 Howard Hughes Medical Institute, Yale University, New Haven, CT, 06520, USA.

Abstract

Background

Regulatory T cells (Tregs) are essential for suppressing inflammation and maintaining tissue homeostasis. The TGF-? signaling pathway plays a fundamental role in Treg differentiation, function, and stability. However, the molecular mechanisms by which TGF-? regulates these processes remain incompletely understood. TIF1? is a protein known to promote a noncanonical TGF-?/Smad pathway, with the ability of modulating gene expression by its unique ability to binding to the crhomatin; additionally it can modulate protein levels through its ubiquitination ligase activity. This ability to ubiquitinate proteins as well as chromatin reader highlights TIF1? as a key player in cellular processes. Despite its established role in other cells subtypes he function of TIF1? in Tregs has not yet been investigated.

Methods

To investigate the role of TIF1? in Treg cell function, we generated conditional knockout (cKO) mice using the Cre-Lox system. TIF1? was selectively deleted in T cells by crossing TIF1?-floxed mice with CD4-Cre mice or specifically in Tregs by crossing TIF1?-floxed mice with Foxp3-Cre mice. These mice were further crossed with Foxp3 reporter strains to track Treg expression dynamics and with Rosa26 reporter strains for fate mapping of Tregs lacking TIF1?. We characterized the phenotype of these animals under homeostatic and inflammatory conditions, including models of autoimmune disease and helminth infection, using flow cytometry to assess Treg frequency, stability, and functional phenotype. To complement this, we performed epigenetic and transcriptional profiling of TIF1?-deficient Tregs. Chromatin accessibility was analyzed using ATAC-Seq, while RNA-Seq was used to examine transcriptional changes and identify pathways influenced by the loss of TIF1?.

Results

We demonstrate that TIF1?-deficient Tregs lose their stability and adopt an effector phenotype in response to inflammatory stimuli. In autoimmune models, these Tregs acquire a Th1-like phenotype or transition into pro-inflammatory exTregs, while in helminth infection models, they exhibit a Th2-like phenotype. The loss of Foxp3 in TIF1?-deficient Tregs is a cell-intrinsic process associated with increased proliferation and enhanced glycolytic capacity upon activation. Furthermore, we show that the deletion of TIF1? leads to significant changes in the methylation status and chromatin accessibility of Tregs, highlighting an epigenetic basis for their instability. Mechanistically, we identified the ?-catenin pathway as the critical driver of TIF1?-dependent Treg stability and proliferation under inflammatory conditions.

Conclusions

Our data demonstrates that TIF1? is required for the maintenance of a suppressor phenotype and stability of Treg lymphocytes during inflammatory conditions in vivo and represents a new modulatory pathway to manipulate Treg cells for therapeutic purposes.