*2.1. Aging Up-Regulated Inflammatory Response-Related Genes and Down-Regulated Genes Associated with Cartilage Development and Homeostasis in Mouse Knee Joints*

To determine aging-related molecular changes in healthy (uninjured) knee joints, we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice using RNA-seq. Compared with 10-week-old mice, 974 and 1732 genes were up-regulated and 1006 and 1386 genes were down-regulated in 62-week-old mice and 95-week-old mice, respectively (Figure 1A,B, Table S1). Six-hundred and four genes including 42 arthritis-associated genes (*Mmp3*, *Ccl3*, *Ccr5*, *Csf3*, *Cxcr6*, *Lyz1*, *Il33*, and *Tnfsf15*, among others), 110 genes associated with immune response (*Tlr7*, *Csf3*, *Cd3d*, *Cd4*, *Nlrp1b*, and *Cd8a*, among others) and 47 inflammatory response-related genes (*Ccl5*, *Ccr5*, *Cxcl1*, and *Cxcl5*, among others) were up-regulated in both 62-week-old and 95-week-old old mice compared with 10-week-old mice (Figure S1A, Figure 1C). Of these common up-regulated genes, 143 genes including several key regulators of immune/inflammatory responses such as *Il5ra*, *Il17re*, *Cxcr3*, *Cxcr6*, *Cxcl5*, *Cxcl9*, *Ccl3*, and *Ccl4* had a significantly higher expression in 95-week-old mice compared with 62-week-old mice, suggesting a progressive increase in the levels of inflammatory mediators in the joint with age (Figure 1C,E). Genes down-regulated in both 62-week-old and 95-week-old mice compared with 10-week-old mice included 39 genes associated with cartilage development (*Col2a1*, *Col9a1*, *Sox9*, *Acan*, and *Comp*, among others), 40 genes associated with bone development (*Col1a1*, *Alpl*, *Sparc*, *Bglap*,

and *Runx2*, among others), 35 genes involved in osteoblast differentiation, and 22 genes involved in chondrocyte differentiation (Figure 1D, Figure S1B–D). Several key regulators of cartilage development and homeostasis including *Sox9*, *Col2a1*, and *Acan* had the lowest expression in 95-week-old mice, indicating a reduction in cartilage anabolic responses with age (Figure 1F).

**Figure 1.** Age-related changes in the knee joint gene expression. Genes up- (**A**) and down-regulated (**B**) in 62-week-old (62W) mice and 95-week-old (95W) mice compared with 10-week-old (10W) mice and in 95-week-old mice compared with 62-week-old mice. (**C**) Inflammatory response-related genes up-regulated in both 62-week-old and 95-week-old compared with 10-week-old mice (top 30 genes). (**D**) Cartilage development-associated genes down-regulated in both 62-week-old and 95-week-old compared with 10-week-old mice. (**E**) Examples of inflammatory response genes showing progressive increase with age. (**F**) Key cartilage development-associated genes showing an age-related decrease in expression.
