Integrative transcriptomic and proteomic profiling reveals altered thymocyte development and microenvironment...

Age-related thymic atrophy (ARTA) is a hallmark of immunosenescence, yet the earliest thymocyte developmental checkpoints affected by increasing age and the coordinated molecular programs that drive thymic degeneration remain incompletely defined.

We compared young (1-month-old) and middle-aged (MA, 12-month-old) male ICR mice using thymus weight/index measurement, histopathology, peripheral blood cell analysis, and immunostaining of thymic markers. We further performed RNA-seq and data-dependent acquisition (DDA) proteomics, followed by integrated transcriptomic-proteomic pathway analyses. Finally, we analyzed public human thymus datasets to assess the translational relevance of our findings.

Middle-aged mice exhibited marked thymic involution with reduced thymus weight and thymic index, accompanied by peripheral lymphopenia and reduced peripheral T-cell counts, while myeloid populations (neutrophils and monocytes) increased. Pathological examination revealed lipid droplet accumulation in the thymus of aged mice, along with decreased Ki-67 expression and an increased number of apoptotic cells. Histologically, aged thymuses showed cortical thinning and an indistinct corticomedullary boundary. Reduced cortical CD25 with increased CD44 is suggestive of a possible developmental impediment around the DN1-to-DN2 transition; in parallel, CD3⁺, CD4⁺, and CD8⁺ T cells were reduced in MA mice. Transcriptomics identified broad remodeling (2,084 upregulated and 255 downregulated genes), featuring heightened inflammatory responses, extracellular matrix (ECM)-receptor interaction, and fatty acid metabolism, with suppression of DNA replication-related programs. Proteomics revealed concordant shifts (189 upregulated and 91 downregulated proteins), including enhanced metabolic and ECM-related pathways and reduced DNA replication and T-cell differentiation signatures. Integrated multi-omics highlighted 289 synchronously upregulated gene-protein pairs enriched in focal adhesion, PI3K/Akt signaling, ECM-receptor interaction, and complement/coagulation cascades, indicating coordinated microenvironmental injury and remodeling during thymic atrophy. In the translational relevance analysis, the aging human thymus exhibited features similar to those observed in mice, including impaired DNA replication, increased ECM-receptor interaction, and enhanced fatty acid metabolism-related activity, with thymic stromal cell analysis indicating that these processes are closely associated with mesenchymal cells.

Increasing age disrupts early thymocyte differentiation and is accompanied by inflammatory-ECM remodeling and adipose-associated metabolic reprogramming. These integrative omics signatures nominate candidate pathways and regulators for developing interventions to mitigate ARTA and preserve immune homeostasis.