Aged skin exacerbates experimental osteoarthritis via enhanced IL-36R signaling

Domain: biology
Published: 2026-01-14
Editorial outcome: major_revision

Abstract
Age-related inflammation plays a pivotal role in osteoarthritis (OA) pathogenesis, but the mechanism is not fully understood. Here, we identify decreased IL-36 receptor antagonists (IL-36Ra) in epidermal keratinocytes from a premature-aged skin mice model, aged mice and patients. Decreased IL-36Ra leads to increased secretion of IL-36 agonists to serum and joints, which activates proinflammatory signaling and promotes senescence in chondrocytes and synovial fibroblasts, thereby aggravates OA progression. Deletion of IL-36Ra in keratinocytes exacerbates, whereas intra-articular inhibition of IL-36R signaling effectively attenuates OA progression in male mice. Moreover, we also generate microneedles loaded with mouse recombinant IL-36Ra protein or spesolimab, insert them directly into skin to sustainably inhibit IL-36R signaling, which both clearly attenuate OA progression in male mice. Overall, our results reveal that IL-36 agonists are age-related systemic inflammatory factors released from skin to joints and contribute to OA development, and targeting IL-36R signaling in aged skin with microneedles represents a promising disease-modifying approach.

Introduction
Osteoarthritis (OA) is a prevalent, slowly degenerative joint disease that causes joint tenderness, stiffness, dysfunction, and pain in the elderly population, affecting quality of life and increasing mortality, with no available licensed disease-modifying treatments1. OA has long been considered as a “wear and tear” disease affecting articular cartilage, but current opinions suggest that inflammation plays a prominent role in OA pathogenesis, leading to a renewed definition of OA as a complex multifactorial joint pathology2. Inflammation in OA has unique characteristics compared to rheumatoid arthritis and other autoimmune diseases, including that it is chronic and associated with low-grade systemic and local inflammation, with involvement of both the innate and adaptive immune responses2–4. Research is increasingly uncovering how age-related systemic and local inflammation can contribute to OA joint damage5–7. Thus, given the pivotal role of low-grade inflammation in OA, future work is still needed to gain a deeper mechanistic understanding of the inflammation, identify novel targets, and develop effective disease-modifying drugs to prevent OA progression.

Skin is the largest organ of the human body, serving as the organismal barrier. However, the skin is fragile and possesses a senescent and inflammatory phenotype when continually exposed to external stressors such as ultraviolet rays and pollution8. The impairment of barrier function in the aged epidermis leads to inflammation of the skin and affects other systems throughout the body9. Recent studies have shown that epidermal barrier dysfunction in aged mice increases the levels of proinflammatory cytokines in the skin and serum, whereas correction of epidermal function in aged mice normalizes both10–13. The joints can be affected by systemic inflammation from skin disease; indeed, 20–30% of patients with psoriasis will develop psoriatic arthritis (PsA), while cytokine inhibition used to control psoriatic skin disease has been shown a protective effect on the risk of PsA, potentially providing a link between the transition of skin-to-joint disease14–16. However, how inflammation of the skin modulates the post-injury inflammatory response in OA has yet to be studied.

The IL-36 subfamily belongs to the IL-1 superfamily and consists of three activating ligands (IL-36α/β/γ), a receptor antagonist (named IL-36Ra in mice and IL-36RN in humans), and their common subfamily-specific receptors (IL-36R and IL-1RAcP). IL-36α/β/γ are agonizts that trigger IL-36R/MyD88 to activate proinflammatory signaling, including nuclear factor (NF)-κB, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)−1/2 intracellular pathways, leading to the production and secretion of inflammatory cytokines, creating a feedback loop17. IL-36Ra mediates its antagonistic effect by binding IL-36R and inhibiting the recruitment of IL-1RAcP, thus truncating the relative signaling pathway. IL-36α/β/γ and IL-36Ra are physiologically present in skin and are mainly produced by keratinocytes, and all IL-36 agonizts have been found to synergize with IL-17 and TNF-α in keratinocytes18. IL-36 cytokines have been implicated in numerous inflammatory diseases, including psoriasis, inflammatory bowel diseases, allergic rhinitis, rheumatoid arthritis, and OA19,20. Recent studies have revealed that IL-36 blockade fails to ameliorate rheumatoid arthritis21,22; intra-articular injection of IL-36Ra or hydrogel can effectively control the inflammatory response, thereby protecting cartilage and slowing the development of OA in mice23–25. However, the origin and targeted cells of IL-36 cytokines involved in OA are not fully elucidated. In this study, we constructed skin aging models in male mice using two approaches: chemical (D-Gal) and genetic (Zmpste24 knockout), and these two specific models can reasonably reflect human skin aging26,27. We confirmed the link between aged skin and OA progression while demonstrated that aged skin keratinocytes are the main source of IL-36 agonizts, inflammatory mediators that promote OA progression. We also showed how best to target the inflammatory response with sustained-release of formulations, which represented a future perspective for clinical anti-inflammatory therapeutic interventions for OA. Together, our data confirm the connection between aged skin and traumatic OA and suggest that premature skin aging may play a crucial role in promoting the progression of traumatic OA in mice through skin–serum–joint crosstalk.

Results
Premature skin aging promotes traumatic OA progression in mice
Age is the strongest risk factor for OA. To explore whether skin aging would affect the development of traumatic OA through skin-to-joint crosstalk, we constructed a mouse model of prematurely aged skin combined with DMM surgery-induced OA. First, we induced premature aged skin in C57BL6/J mice by cutaneous application of D-galactose (D-gal), which did not increase the systemic level of D-gal as we reported earlier (Supplementary Fig. 1a)28. DMM surgery was performed in these mice, and in one group of mice, the aged skin was removed at the same time as DMM surgery (Fig. 1a). Premature skin aging was successfully induced in D-gal-treated mice, which displayed a thinner epidermis, decreased cell numbers, and immunofluorescence staining revealed an increased number of p16-positive keratinocytes and a decreased Lamin B1 expression, both of which are markers of cellular senescence (Fig. 1b, c). We also observed more cartilage degradation, synovitis, subchondral bone sclerosis, and osteophyte development in D-gal-treated mice at 2 months after DMM surgery. Remarkably, compared to D-gal-treated mice, aged skin-removed mice displayed only a minor loss in proteoglycan content, with thicker cartilage, decreased synovitis, less osteophyte formation, and lower OARSI scores (Fig. 1d, e). Immunostaining showed increased expression of the catabolic marker metalloproteinase 13 (MMP13) and the fibrosis and lymphatic angiogenesis marker podoplanin (PDPN), along with decreased expression of the anabolic marker Collagen type II (Col2a1) in D-gal-treated mice, while the opposite was observed in skin-removed mice (Fig. 1f, g). Furthermore, the Von Frey filament test verified enhanced knee joint pain in D-gal-treated mice, which was recovered in skin-removed mice (Fig. 1h). Studies in aged mice have claimed that epidermal-barrier dysfunction increases the levels of proinflammatory cytokines in the skin and serum10. To further demonstrate the correlation between skin aging and OA, we established DMM-induced OA in C57BL/6J mice and administered weekly intra-articular injections of serum from normal mice or mice with D-Gal cutaneous application (Fig. 1i). Two months after DMM surgery, cartilages from mice treated with serum from skin-aged mice displayed more severe cartilage erosion and clefts, whereas those from mice treated with serum from normal mice were comparable to controls (Fig. 1j, k). The Von Frey filament test confirmed consistent levels of pain in both model types (Fig. 1l). These data indicate that mice with premature skin aging may accelerate OA progression through serum components.

Next, we generated mice with Zmpste24 conditional knockout (Z24-cKO) in epidermal keratinocytes to induce skin premature aging via topical administration of capsid-mutant AAV vectors encoding Cre recombinase (AAV2-Cre) in Zmpste24fl/fl mice26. We confirmed high deletion efficiency of Zmpste24 in epidermal keratinocytes but not in other organs, and compared to control mice, the skin of Z24-cKO mice showed a thinner epidermis, decreased cell numbers, loss of rete ridges, and remarkedly increased senescent keratinocytes, manifested as an increase in p16 and a decrease in Lamin A/C expression, a nuclear envelope stability protein whose processing is impaired due to Zmpste24 knockout (Supplementary Fig. 1b–f). Zmpste24fl/fl and Z24-cKO mice were then subjected to DMM surgery to induce OA, as described in the schematic (Supplementary Fig. 2a). Interestingly, 2 months after DMM surgery, we noticed uneven cartilage surface and loss of proteoglycan content in Zmpste24fl/fl mice, whereas Z24-cKO mice displayed more severe cartilage erosion and even clefts. Z24-cKO mice also exhibited more severe synovitis, subchondral bone sclerosis, osteophyte development and enhanced knee joint pain (Supplementary Fig. 2b–d). The immunostaining results showed enhanced matrix MMP13 and PDPN and decreased Col2a1 expression in Z24-cKO mice, indicating a degenerative process in the cartilage and enhanced synovial inflammation (Supplementary Fig. 2e, f). Together, our data confirm the connection between aged skin and traumatic OA and suggest that premature skin aging may play a crucial role in promoting the progression of traumatic OA in mice through skin–serum–joint crosstalk.

Decreased IL-36Ra in senescent keratinocytes results in increased systemic IL-36 agonizts and is involved in OA
To elucidate the underlying reasons of aged skin in promoting traumatic OA progression, we performed proteomic screen analysis with epidermal skin tissue from young (3 months old) and aged (18 months old) cutaneous application mice (Supplementary Table 1). As aged skin is fragile and possesses a senescent and inflammatory phenotype, a downregulated protein, IL-36Ra attracted our attention (Fig. 2a). IL-36Ra, a receptor antagonist of the IL-36 subfamily, suppresses inflammatory responses by competitively binding to IL-36R, and is mainly produced by keratinocytes in skin and was reported to be associated with the progression of OA24. In aged mice, the skin displayed a thinner epidermis, decreased cell numbers, and increased senescent keratinocytes (Fig. 2b, c), whereas joint cartilages showed fibrillation and loss of proteoglycan content (Fig. 2d). Immunostaining of the IL-36 family and co-staining with keratin 10 (K10), a type I keratin expressed in suprabasal keratinocytes of the skin, confirmed that IL-36Ra is specifically expressed in epidermal keratinocytes and decreased in senescent keratinocytes of aged skin. In contrast, IL-36 activating ligands (IL-36α/β/γ) are enhanced in senescent keratinocytes, serum, and joint tissues (Fig. 2e–h). We next observed a decrease in the expression of IL-36Ra and an increase in the expression of IL-36 activating ligands in D-Gal-treated mouse skin keratinocytes (Supplementary Fig. 3a–c). IL-36 activating ligands were elevated in the serum of D-Gal mice, but decreased after the removal of D-Gal from the skin (Supplementary Fig. 3d). Moreover, upregulated mRNA expression of IL-36 activating ligands was also detected in left joints from mice treated with D-Gal (Supplementary Fig. 3e). Decreased IL-36Ra in keratinocytes and increased IL-36 agonizts in keratinocytes, serum, and joint tissues were also observed in Z24-cKO mice compared to Zmpste24fl/fl mice (Supplementary Fig. 3f–i). Furthermore, we extracted and cultured primary keratinocytes from young mice and induced senescence by UVB irradiation. Immunostaining and western blot analysis confirmed successfully induced senescence, which was accompanied by decreased IL-36Ra in cultured keratinocytes (Supplementary Fig. 4a–c), and increased IL-36 agonizts in the supernatant liquor (SNL) via ELISA analysis (Supplementary Fig. 4d). Next, we collected epidermal skin from young patients without OA (35 ± 5 years old) and old patients with OA (70 ± 5 years old). Compared to young patients, an increased number of senescent keratinocytes in the skin epidermis and enhanced degradation of proteoglycan content in cartilage were observed in aged patients (Fig. 2i–k). In addition, diminished expression of IL-36RN in keratinocytes and elevated expression of IL-36 agonizts in keratinocytes and serum were detected in aged patients, as we detected previously with cytokine antibody array28 (Fig. 2l–n and Supplementary Fig. 4e). We also found positive correlations of elevated expression of IL-36 agonizts between serum and synovial fluid in aged OA patients, with IL-36α concentrations 359.27 ± 21.78 pg/mL in serum and 337.01 ± 15.36 pg/mL in synovial fluid, IL-36β concentrations 376.32 ± 18.83 pg/mL in serum and 361.26 ± 18.97 pg/mL in synovial fluid, IL-36γ concentrations 546.15 ± 35.58 pg/mL in serum and 535.12 ± 10.25 pg/mL in synovial fluid. The determination coefficients (R²) of IL-36α, β, and γ were 0.4716, 0.4660, and 0.3210, respectively (Fig. 2o). After inducing senescence in human keratinocyte HaCat cells with UVB irradiation, we observed decreased IL-36RN and increased IL-36 agonizts in the SNL (Supplementary Fig. 4f–i). These data verified that decreased IL-36Ra in senescent keratinocytes from aged skin results in increased systemic IL-36 agonizts and maybe involved in OA.

IL-36 isoforms are widely expressed in many tissues17. To determine whether skin is the predominant site of IL-36Ra expression and whether the expression is changed with aging, we detected IL-36Ra expression in all reported tissues from young and aged mice. IL-36Ra expression was observed in almost all tissues and organs we analyzed, showing the highest expression in epidermal skin in young mice (Supplementary Fig. 5a). In aged mice, its expression was increased in the spleen but markedly decreased in the epidermal skin (Supplementary Fig. 5b, c). We also noticed lower expression of IL-36Ra in the cartilage and synovium tissues from aged mice, as reported previously. Further analysis with tissues from more age groups revealed that the reduction in IL-36Ra occurred earlier in the skin than in the articular cartilage and synovium (Supplementary Fig. 5d, e). In addition, skin has greater volume and weight than other tissues. We therefore conclude that decreased IL-36Ra in senescent keratinocytes from aged skin is responsible for most systemic increases in IL-36 agonizts and is involved in OA progression.

IL-36Ra deletion in the epidermis keratinocytes activates proinflammatory signaling in vivo
Next, we generated epidermis keratinocyte conditional knockout mice (IL-36Ra-cKO) with topical administration of capsid-mutant Adeno-associated virus 2 (AAV2) vector23 encoding Cre recombinase (AAV2-Cre) to IL-36Rafl/fl mice. Pathological histology stainings showed that the epidermis did not differ significantly between young IL-36Rafl/fl and IL-36Ra-cKO mice (Fig. 3a, b). Effective deletion of IL-36Ra was confirmed in epidermal keratinocytes but not in other internal organs of IL-36Ra-cKO mice via immunostaining and western blot analysis, and enhanced IL-36 agonizts were observed in keratinocytes of IL-36Ra-cKO mice (Fig. 3c, d and Supplementary Fig. 6a–c). Moreover, the expression level of IL-36 agonizts in serum and joint sections from IL-36Ra-cKO mice was significantly increased compared to that in IL-36Rafl/fl mice (Fig. 3e, f), confirming that deletion of IL-36Ra in keratinocytes was responsible for systemic increase in IL-36 agonizts. Subsequently, epidermal skin tissues were collected from IL-36Rafl/fl and IL-36Ra-cKO mice for RNA sequencing analysis. KEGG pathway enrichment and gene set enrichment analysis (GSEA) showed that, compared to IL-36Rafl/fl mice, IL-36Ra conditional knockout in keratinocytes resulted in activation of downstream pathways, including NF-κB, MAPK and JAK-STAT signaling, as previously reported (Fig. 3g–i). We noticed elevated expression of key factors in these pathways, including p-p65, p-STAT3, p-p38, p-JNK and p-ERK1/2 in epidermis keratinocytes of IL-36Ra-cKO mice by immunostaining (Fig. 3j, k), confirming that ablation of IL-36Ra results in the activation of proinflammatory signaling in the epidermis. Further, we also detected activated NF-κB, MAPK and JAK-STAT signaling in cartilage and synovium from IL-36Ra-cKO mice, compared with IL-36Rafl/fl mice (Fig. 3l, m). After all, these data demonstrate that IL-36Ra deletion in the epidermis keratinocytes increases systemic IL-36 agonizts and results in activation of proinflammatory signaling in skin and joint, including NF-κB/JAK-STAT/MAPK pathways.

IL-36Ra ablation in the epidermis keratinocytes aggravates experimental and age-related OA development in mice
IL-36Rafl/fl and IL-36Ra-cKO mice were then subjected to DMM surgery to induce OA at 12-week age, as described in the schematic (Fig. 4a). Importantly, at 2 months after DMM surgery, safranin O staining revealed that IL-36Ra-cKO mice displayed greater cartilage degradation, synovitis, subchondral bone sclerosis, osteophyte maturity, and markedly increased OARSI scores than IL-36Rafl/fl mice. However, no significant differences in SBP thickness were observed between IL-36Ra-cKO and IL-36Rafl/fl mice (Fig. 4b, c). Further analysis showed increased MMP13, PDPN, and p16 expression, decreased Col2a1 expression (Fig. 4d, e), and enhanced knee joint pain (Fig. 4f) in IL-36Ra-cKO mice, indicating that IL-36Ra ablation in the epidermal skin promotes experimental OA pathogenesis in mice. Furthermore, the histopathological changes of the epidermal skin and joints in IL-36Rafl/fl and IL-36Ra-cKO mice without surgical intervention were analyzed at 10 months of age. We did not notice serious psoriasis-like lesions in the epidermal skins of IL-36Ra-cKO mice29, but thinner epidermis, lower cell numbers, and decreased epidermal acanthosis were observed compared to IL-36Rafl/fl mice (Fig. 4g). IL-36Ra-cKO mice exhibited increased cartilage destruction and aggravated synovitis in the joints (Fig. 4h, i). Immunostaining confirmed an imbalance in cartilage metabolism and enhanced chondrocyte senescence (Supplementary Fig. 6d, e), and the Von Frey filament test confirmed enhanced knee joint pain (Fig. 4j) in IL-36Ra-cKO mice. Thus, our data identify the vital role of epidermal skin IL-36R signaling in OA, IL-36Ra ablation in the epidermis keratinocytes aggravates experimental and age-related OA development in mice.

IL-36 agonizts from keratinocytes induce chondrocytes catabolism and inflammation in synovial fibroblasts
To determine which cells are targeted by IL-36 agonizts, we next performed IL-36R immunostaining in various key tissues and cells involved in OA from mice and human joint sections at a young and old age. In the synovium, we observed gradual increases in IL-36R-positive cells with age, and using specific markers, we confirmed that the majority of IL-36R positive cells were synovial fibroblasts, as indicated by PDPN expression, while no significant co-localization was observed with F4/80-positive macrophages (Fig. 5a). We also noticed that IL-36R was positive in articular cartilage chondrocytes, and that the number of positive chondrocytes reached a peak in mice at middle age (10 months), before decreasing at old age (18 months) (Fig. 5b). Moreover, similar changes in IL-36R expression were observed in chondrocytes and synovial fibroblasts from young and aged humans (Supplementary Fig. 7a, b). Furthermore, we detected more blood vessels (CD31) but decreased lymph vessels (PDPN) in the synovium of aged and OA mice (Supplementary Fig. 7c, d), indicating that systemic IL-36 agonizts may affect chondrocytes and synovial fibroblasts via vascular vessels in the synovium. Subsequently, we cultured mouse keratinocytes and induced senescence with UVB irradiation. We next investigated the effects of supernatant liquor from aging keratinocytes (Aging SNL) on chondrocytes or synovial fibroblasts with mIL-36Ra or IL-36R knockdown (Fig. 5c). For synovial fibroblasts, the migration ability, cellular senescence, and release of inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β) were significantly enhanced under the stimulation of Aging SNL, but were restored to levels equal to those of the control group after the addition of mIL-36Ra or knockdown of IL-36R (Fig. 5d, e). Furthermore, in cultured mouse primary chondrocytes, loss of proteoglycan content, increased cellular senescence, inhibited anabolism, and enhanced catabolism were noticed upon stimulation with Aging SNL, all of which were recovered by mIL-36Ra and IL-36R knockdown (Fig. 5f, g). This suggests that the Aging SNL induces catabolic processes in chondrocytes and inflammatory responses in synovial fibroblasts through the IL-36R signaling pathway.

RNA-sequencing analysis was then performed in the negative control, and chondrocytes stimulated with the Aging SNL groups. KEGG and GSEA analysis showed that, compared to the control group, IL-36-related signaling, including NF-κB, JAK-STAT and MAPK signaling, were activated in chondrocytes under the stimulation of Aging SNL (Supplementary Fig. 8a). We confirmed the upregulation of p-p38, p-ERK1/2, p-JNK, p-p65, and p-STAT3 in both chondrocytes and synovial fibroblasts following stimulation with Aging SNL, which were recovered after addition of mIL-36Ra (Supplementary Fig. 8b). To confirm that IL-36Ra reduction is critical for the function of senescent keratinocytes, we knocked down IL-36Ra with small interfering RNA in cultured mouse keratinocytes, and used the resulting supernatants to stimulate synovial fibroblasts and chondrocytes (Fig. 5h). Interestingly, we observed similar changes, with increased inflammation in synovial fibroblasts and chondrocyte catabolism with the stimulation of supernatant liquor from IL-36Ra knockdown keratinocytes (IL-36Ra-KD SNL), both of which were recovered by mIL-36Ra and IL-36R knockdown (Fig. 5i–l and Supplementary Fig. 8c). Moreover, activation of these signaling pathways was also verified in joint sections from mice with prematurely aged skin with Immunostaining (Supplementary Fig. 8d–f). Thus, these data suggest that loss of IL-36Ra in keratinocytes leads to increased IL-36 agonist secretion and induces chondrocytes catabolism and synovial inflammation by activating pro-inflammatory signaling, including NF-κB/JAK-STAT/MAPK pathways.

Insertion mIL-36Ra-loaded microneedles into skin targeting epidermal keratinocytes attenuates experimental OA in mice
To further demonstrate the effectiveness of targeted IL-36R signaling in preventing OA in vivo, we established DMM-induced OA and used pharmacological and genetic approaches to inhibit IL-36R in control, Z24-cKO mice (Supplementary Fig. 9a). Mice received intra-articular delivery of mIL-36Ra or IL-36R knockout AAV (shIL-36R) weekly, neither of which affected the normal appearance of the articular cartilage in the knee joints of sham-operated mice (Supplementary Fig. 9b). In Zmpste24fl/fl and Z24-cKO mice with DMM-induced post-traumatic OA, Z24-cKO mice exhibited more severe cartilage destruction, subchondral bone sclerosis, osteophyte development, and synovial inflammation. mIL-36Ra and IL-36R knockout treatment significantly improved the OA-relevant symptoms both in Zmpste24fl/fl and Z24-cKO mice, and mIL-36Ra treatment even restored the degree of OA in Z24-cKO mice to a level comparable to that in Zmpste24fl/fl mice with similar OARSI structure scores (Supplementary Fig. 9c, d). Detection of cartilage metabolism confirmed the upregulated anabolic factors and downregulated catabolic factors, the inhibition of synovial inflammation by mIL-36Ra treatment and IL-36R knockout, and the alleviation in knee joint pain in both groups (Supplementary Fig. 9e–g). We further validated the ameliorating effect of intra-articular delivery mIL-36Ra or IL-36R knockout AAV on changes in OA pathogenesis and pain in IL-36Rafl/fl and IL-36Ra-cKO mice (Supplementary Fig. 10a). Importantly, the use of mIL-36Ra also showed a better effect than IL-36R knockout in IL-36Ra-cKO mice (Supplementary Fig. 10b–g). Collectively, our data suggest that intra-articular knockout of IL-36R or mIL-36Ra delivery to inhibit IL-36R signaling significantly ameliorates OA pathology changes in mice, including wild-type mice, mice with premature aged skin, and mice with IL-36Ra ablation in the epidermis.

In this study, we reached a similar conclusion to previous studies, in that IL-36Ra is a potential therapeutic agent to ameliorate OA23. We also demonstrated that senescent keratinocytes in aged skin represent the major source of IL-36 agonizts and promote OA progression. However, given that weekly or daily injections are impractical for clinical use, we developed MN-mIL-36Ra, microneedles loaded with mIL-36Ra to achieve sustained release of mIL-36Ra, and inserted them directly into the skin to target epidermal keratinocytes (Fig. 6a, b). The images of MN-mIL-36Ra under optical and scanning electron microscopy (SEM) are displayed in Fig. 6c. The in vitro drug release profile showed that the drug-release rate of mIL-36Ra was 41.06% within 24 h, 73.37% within 168 h, indicating that MN-mIL-36Ra had a sustained release effect (Fig. 6d). Next, we established DMM-induced OA in control, Z24-cKO, or IL-36Ra-cKO mice, and covered the skin of mice with MN-mIL-36Ra to examine its effect on OA protection (Fig. 6e). Upon application of MN-mIL-36Ra in mice, the skin exhibited rapid recovery within minutes, skin epidermis and other important organs were not significantly affected after 8 weeks (Supplementary Fig. 11a–d). Reduced IL-36 ligands in keratinocytes and serum were observed in Z24-cKO and IL-36Ra-cKO mice with MN-mIL-36Ra application, while IL-36Ra levels in serum were not affected (Fig. 6f–h). SOFG staining demonstrated that MN-mIL-36Ra treatment effectively improved cartilage histological structural integrity relative to the control group both in Zmpste24fl/fl and Z24-cKO mice, and antagonized the enhanced OA pathology changes and knee joint pain caused by premature skin aging in Z24-cKO mice (Supplementary Fig. 11e and Fig. 6i–k). Moreover, chondrocyte metabolism and synovial inflammation improved and recovered to levels similar to those of Zmpste24fl/fl in Z24-cKO mice (Supplementary Fig. 11f-g). MN-mIL-36Ra treatment also showed comparable alleviation of all OA symptoms in IL-36Rafl/fl and IL-36Ra-cKO mice, including cartilage and subchondral bone structures, synovitis, osteophyte hyperplasia, chondrocyte metabolism, and knee joint pain (Supplementary Fig. 11h–j, Fig. 6l–n). Thus, MN-mIL-36Ra provides an efficient strategy for the treatment of OA, directly targeting IL-36 agonizts derived from skin keratinocytes and provides a promising drug delivery system to broaden the usage of mIL-36Ra.

Targeting IL-36R signaling demonstrates potential therapeutic efficiency for human OA
Spesolimab is an IL-36R antagonist approved by the Food and Drug Administration for the treatment of generalized pustular psoriasis flares in adults30,31. We next assessed the potential clinical therapeutic efficacy of spesolimab in human OA using the human C28/I2 chondrocyte cell line, human fibroblast-like synoviocytes, primary human articular chondrocytes, and cartilage tissue explants from patients undergoing total knee arthroplasty. C28/I2 cells were treated with SNL from aging HaCat cells or HaCat cells with IL-36RN knockdown. Loss of chondrocyte metabolism homeostasis and proteoglycan content were determined by immunoblotting and alcian blue staining. The addition of spesolimab to C28/I2 cells showed a good protective effect against the SNL from both HaCat cells (Fig. 7a and Supplementary Fig. 12a). Similar results were obtained in primary human articular chondrocytes from patients with OA, and spesolimab was found to significantly retain the anabolism and proteoglycan content (Fig. 7b). In cartilage tissue explants from patients with OA cultured with spesolimab for 14 days, the degradation of proteoglycan content was inhibited, the expression of degradative enzymes was reduced, and the expression of anabolic factors was increased, indicating the therapeutic efficacy on OA pathogenesis (Fig. 7c, d). In human fibroblast-like synoviocytes, spesolimab treatment effectively reduced cell migration, suppressed cellular senescence, and led to decreased release of inflammatory factors (Fig. 7e, f).

Spesolimab is a humanized IgG1 monoclonal antibody against human IL-36R and previous study claimed that spesolimab has low binding affinity to IL-36R in other species up to 1μM32. We subsequently performed an SPR assay and confirmed spesolimab had low binding affinity with mouse IL-36R at low concentration, but had higher affinity at higher concentrations, with a KD value of 5.34 × 10⁻⁶ M, a Ka value of 4.92 × 10⁴ 1/Ms, and a Kd value of 2.62 × 10⁻¹ 1/s (Supplementary Fig. 12b). We investigated the effects of Aging SNL on chondrocytes or synovial fibroblasts with spesolimab. Interestingly, we observed similar changes, with increased inflammation in synovial fibroblasts, activation of pro-inflammatory signaling in chondrocytes and synovial fibroblasts, as well as chondrocyte catabolism with the stimulation of Aging SNL, all of which were recovered by spesolimab (Fig. 7g–j and Supplementary Fig. 12c). Next, we established DMM-induced OA in control, Z24-cKO, or IL-36Ra-cKO mice, and covered the skin of mice with MN-SPE (microneedles loaded with spesolimab) to examine its effect on OA protection (Fig. 7k). Upon application of MN-SPE in mice, skin epidermis and other important organs were not significantly affected after 8 weeks (Supplementary Fig. 12d–f). Reduced IL-36 ligands in keratinocytes and serum were observed in Z24-cKO and IL-36Ra-cKO mice with MN-SPE application, while spesolimab levels in serum were not affected (Supplementary Fig. 12g–i). SOFG staining demonstrated that MN-SPE treatment effectively improved cartilage histological structural integrity relative to the control group, both in Zmpste24fl/fl and Z24-cKO mice, and antagonized the enhanced OA pathology changes and knee joint pain caused by premature skin aging in Z24-cKO mice (Fig. 7l–m, Supplementary Fig. 12j–l). MN-SPE treatment also showed comparable alleviation of all OA symptoms in IL-36Rafl/fl and IL-36Ra-cKO mice, including cartilage and subchondral bone structures, synovitis, osteophyte hyperplasia, chondrocyte metabolism, and knee joint pain (Fig. 7n, o and Supplementary Fig. 12m–o). Thus, MN-SPE provides an efficient strategy for the treatment of OA, directly targeting IL-36 agonizts derived from skin keratinocytes and provides a promising drug delivery system to broaden the usage of spesolimab.

Methods
Humansamples
Humanskinsamplesfromyoung(30-40yearsold;n=6;halfmaleandhalf female) and elderly (70-75 years old; n=6; half male and half female) individualswereobtainedfromthebackincisionsofpatients undergoingposteriorspinalsurgery.HumanOAcartilageandsyno- vium were taken from patients with late-stage OA undergoing total knee arthroplasty (n=8; aged 60–90; half male and half female). Humannon-OAcartilageandsynoviumwerefrompatientswithbone tumorsundergoingpartialkneereplacementsurgerywithnohistory ofOA(n=6;aged25–45;halfmaleandhalffemale).HumanOAjoint synovialfluidandserumwerecollectedfromthesamepatientswith late-stageOAundergoingtotalkneereplacementsurgery(n=15;aged 70–90;males).Humanserumfromyoung(n=5;aged25–35;2male and3female)andelderly(n=5;aged60–80;2maleand3female) individualswasobtainedfromthehospitalhealthexaminationcenter. All clinical samples for this project were collected from the Third AffiliatedHospitalofSouthernMedicalUniversity(Guangzhou,China), withapprovalfromthehospital’smedicalethicscommittee.Written informedconsentwasobtainedfromallsubjectsortheirguardians, includingconsenttopublishpotentiallyidentifyinginformation.

Mice
All mice were housed in the Specific Pathogen-Free (SPF) animal facility at SouthernMedicalUniversity (Guangzhou,China),with no morethanfivemicepercage.Themicewerekeptinanenvironment withadequateventilation,appropriatetemperatureandhumidity,and hadfreeaccesstofoodandwater.A12hlight/darkcyclewasmain- tained.Allanimalexperimentsconductedinthisstudywereapproved bytheAnimalCareandUseCommitteeofSouthernMedicalUniversity andwereincompliancewithallrelevantethicalregulationsregarding animal research. Twelve-week-old male C57BL/6J mice used forthe experimentalOA and 3-, 10- and 18-month-old male C57BL/6J mice usedforaged-OAstudieswereallacquiredfromtheLaboratoryAnimal CenterofSouthernMedicalUniversity.Zmpste24-floxmiceandIl1f5(IL- 36Ra)-flox mice were purchased from GemPharmatech Co., Ltd (Nanjing, China). Adeno-associated viruses (AAV2/Kera2-Cre-Null- ZsGreenorAAV2/Kera2-ZsGreenControl)wereengineeredbyHanbio BiotechnologyCo.,Ltd(Shanghai,China).WedilutedAAV2(1.4×10^12 vg/mL,20μl)withPBSanduniformlyappliedittotheshaveddorsal skin area (4.0×2.0cm²) of mice once a week. Male mice with Zmpste24-floxandIL-36Ra-floxbackskininfectedbyAAV2/Kera2-Cre- Null-ZsGreen were designated as Zmpste24-cKO and IL-36Ra-cKO, respectively,whilethoseinfectedwithAAV2/Kera2-ZsGreenControl onthebackskinofZmpste24fl/flandIL-36Rafl/flmalemiceweredesig- natedascontrolsforZmpste24-cKOandIL-36Ra-cKO.

Animalsmodel
To induce premature skin aging in mice, D-Galactose (D-Gal; Med- ChemExpress,Cat.No.HY-N0210)wasdissolvedin70%ethanolata concentration of 20mg/mL to facilitate dermal absorption. Then, 200μLofthesolutionwastopicallyappliedoncedailyontoashaved dorsal skin area (4.0×2.0cm²) of 8-week-old male mice for two months.Thecontrolgroupwasonlytreatedwith70%ethanol.Afterone monthofD-Galapplication,weperformedaskinexcisionsurgeryunder sterile conditions. Briefly, after anesthetizing the mice, the shaved dorsalskinwasdisinfectedwithiodine.Usingsterilescissorsandfor- ceps,wecarefullyexcisedtheD-Gal-treatedskinalongtheedgesofthe treatmentarea.Thewoundedgeswerethensuturedusingabsorbable sutures(Vicryl,5-0),followedbyasecondroundofiodinedisinfection. Aftersuturing,thewoundwascoveredwithsterilegauzeandsecured withanelasticbandage.Forthefirstweekpost-surgery,thewoundwas disinfected daily, and the sterile gauze was replaced. Additionally, enrofloxacin(50–100mg/L)wasadministeredinthedrinkingwaterto prevent infection, continuing for 1-2 weeks post-surgery or until the woundhadfullyhealed.ForD-Gal-inducedprematureskinaging,D-Gal skinremoval,andcontrolC57BL/6Jmalemice,aswellasZmpste24-cKO, IL-36Ra-cKOmalemice,andtheirrespectivecontrols(Zmpste24fl/fland IL-36Rafl/fl male mice), we induced OA through Destabilization of the MedialMeniscus(DMM).Briefly,afteranesthetizingthemice,theright kneewaspreparedanddisinfected.Thejointcapsuleadjacenttothe medial side of the patellar tendon was opened to expose the inter- condylar area for visualization and transection of the meniscotibial ligament.Thejointcapsuleandskinwerethenclosed.Forthesham- operatedgroup,thejointcapsulewasopened,butthemeniscotibial ligamentwasnottransected.Ateightweekspost-operation,micewere euthanizedbyrapidcervicaldislocation,andserum,kneejoints,and dorsalskinsampleswerecollected.Forthetransgenicmice(Zmpste24- cKO,IL-36Ra-cKO),weimmobilizedandexposedtheirrightlegs,then administeredan8μlinjectionofIL-36Rantagonistadenovirusorcon- trolvirus(providedbyHanbioBiotechnologyCo.,Ltd.)intothejoint capsuletoknockoutIL-36R,ora10μl(0.5mg/mL)injectionofmouse recombinant IL-36Ra protein (mIL-36Ra, TargetMol, Cat No. TMPY- 04275) to inhibit IL-36R. A booster dose was administered weekly thereafter to maintain the viral load. For C57BL/6J male mice, we injected10μlofserum(sourcedfromD-Gal-inducedprematureskin aging and control mice) into the joint capsule, with injections main- tainedonaweeklybasis.Allmicesubjectedtointra-articularinjections commencedtreatmentoneweekafterDMMsurgeryandwereeutha- nizedatweek8post-surgeryforthecollectionofkneejointsamples.All proceduresstartedoneweekpost-DMMsurgeryandcontinueduntil week8post-OAinduction,atwhichpointmicewereeuthanized,and skinandkneejointsampleswerecollected.

PreparationandcharacterizationofMN-mIL-36RaandMN-SPE
To address the challenges of low oral administration efficiencyand poorcompliancewithinjectablemedications,wedevelopedadissol- vingmicroneedle(DMN)deliverysystemloadedwithmIL-36Ra(Tar- getMol,CatNo.TMPY-04275)orspesolimab(MedChemExpress,Cat No. HY-P99396) forthe treatment of OA. mIL-36Ra was formulated into a gel-based microneedle (MN) system as MN-mIL-36Ra, with a dosageof0.05mgperpatch.Briefly,wefirstuniformlymixed2%(w/v) HyaluronicAcidMethacryloyl(HAMA,EACINBIO;productnumberYX- HM-231121-01-H),0.25%(w/v)LithiumPhenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP, EFL; product number 85073-19-4), and mIL-36Ra (<1.5%)indeionizedwaterina37°Cwaterbathtoensureevendis- tributionofmIL-36RaintheHAMA.TheHAMA-mIL-36Ramixturewas used as the aqueous phase, while the oil phase served as the con- tinuous phase. These were injected into microfluidic chip channels (channelsize:20-50μm)toformstabledropletsoftheaqueousphase intheoilphase.Subsequently,thedropletsweresolidifiedintomIL- 36Ra-loadedmicrospheresbyUVpolymerization.Next,weprepareda 5%(w/v)GelatinMethacryloyl(GelMA,EFL;modelEF-GM-90)solution bydissolving250mgofGelMAand12.5mgofLAPin5mlofdeionized waterat37°Cinawaterbath.WemixedtheGelMAsolutionwiththe mIL-36Ramicrospheres,thenaddedthismixturetothemold,covering themicroneedletips.Themoldwasplacedina60°Cdryingovento removeexcessairbubbles,followedbyUVpolymerizationtoformthe microneedletips.Afterward, a sufficient amount of GelMA solution withoutmIL-36Rawasaddedtoformthebaseofthemicroneedles, andUVpolymerizationwasperformed.Themoldwasfrozenat-20°C forabout30mintoharden,andafterdemolding,theMN-mIL-36Ra microneedleswereobtained.Spesolimabwasformulatedintothegel- basedmicroneedle(MN)systemasMN-SPEwithadosageof0.1mgper patch.Briefly,wefirstpreparedagelbymixing15%(w/v)GelMA,0.25% (w/v) LAP, and 5% (w/v) PEGDA (Sigma Alorich; product number 455008)indeionizedwaterat37°C.Spesolimabwasthenmixedinto thegel.Subsequently,thespesolimab-containinggelwaspouredinto theneedle-partofapolydimethylsiloxane(PDMS)mold,andthemold was placed in a vacuum oven to remove air bubbles, followed by ultraviolet light polymerization. Additional drug-free mixed gel was added to the mold surface as a base, followed by drying and light polymerization until the MN-SPE was demolded. The surface mor- phologyofbothMN-mIL-36RaandMN-SPEwereobservedusingan opticalmicroscope(Leica)andscanningelectronmicroscope(SEM), andtheirinvitrodrugreleaseprofileswereevaluated.Finally,both MN-mIL-36Ra and MN-SPE (microneedle side) were applied to the shavendorsalskin(4×5cm)oftransgenicmalemice(Zmpste24-cKO, IL-36Ra-cKO, Zmpste24fl/fl, and IL-36Rafl/fl), covered with moist gauze, secured with tape to prevent movement, and the moist gauze was changeddaily.

Surfaceplasmonresonance(SPR)analysis
SPRanalysiswasperformed byMedChemExpress(Monmouth Junc- tion, NJ, USA) using a Biacore T200 instrument (GE Healthcare) equipped with CM5 sensor chips (GE Healthcare). Briefly, the chip surface was activated by injection of amixturecontaining1-ethyl-3-(3- dimethylaminopropyl)carbodiimide(EDC)andN-hydroxysuccinimide (NHS)ataflowrateof10μL/min.MouseIL-36R(MedChemExpress; Cat No. HY-HY-P70748) was diluted to 50μg/mL in 10mM sodium acetatebuffer(pH5.0)andimmobilizedontheactivatedsurfaceat 10μl/minfor420s.Thechannelwasblockedwith1Methanolamine (Sigma,CatNo.E6133)(pH8.5)at10μl/minfor420s.Aneighboring channel was activated and blocked similarly but without protein immobilizationtoserveasthereference.Bothchannelswereequili- bratedwithPBS.Spesolimabwasdilutedtoaseriesofconcentrations (0.15625μM,0.3125μM,0.625μM,1.25μM,5μM,10μM)inPBSand flowedoverthechannelsat30μl/minfor150s.Aftereachinjection, thechipwasregeneratedwith10mMglycine-HCl(pH2.0)at10μl/min for5min.DatawerecollectedusingBiacoreT200Controlsoftware, andtheresultsweresubtractedbythosefromthereferencechannel. By globally fitting the data to a 1:1 Langmuir binding model using BiacoreT200Evaluationsoftware,thebindingaffinity(KD),associa- tionrateconstant(Ka),anddissociationrateconstant(Kd)ofspeso- limab with Mouse IL36R were determined. Data were exported to GraphPadPrism9.3softwareforgeneratingthefinalfigures.

VonFery
ToevaluateOA-relatedpainandtheefficacyoftreatmentsinmice,we utilizedthevonFreyfilamentstoassesstheoccurrenceofmechanical allodynia.Attheeighthweekpost-DMMsurgery,micewereplacedina plexiglasschamberwithametalmeshfloor,allowingdirectaccessto theundersideofthemice’sheels.Afterthemicefullyacclimatedtothe setup(atleast15min)andceasedexploratorybehavior,wetestedfor mechanicalallodyniabyapplyingvonFreyfilamentstotheunderside ofthemice’sheelinascendingforceorderforupto5sec.Apositive responsewasindicatedbyasharpretractionofthetestedfoot,and thisvaluewasrecorded.Finally,wecollectedandanalyzedtheaverage ofthreerepeatedtestsforallmice.

Histologicalanalysis
Softtissues,includinghumanandmouseskin,synovium,mouseheart, brain,spleen,lungs,liver,kidneys,intestines,esophagus,stomach,and pancreas,werefixedin4%paraformaldehydefor12h.Humancartilage andmousekneejointswerefixedin4%paraformaldehydefor48h, followedbydecalcificationin10%EDTA(pH8.0)for21days.Alltissues were dehydrated in an automatic dehydration machine (Leica; ASP300S) and then embedded in paraffin. Soft tissues and human cartilageweresectionedcontinuouslyat4μmthickness,whilemouse kneejointsweresectionedcontinuouslyat4μmthicknesswith20μm intervalsaccordingtoastandardprotocol.Allsectionswereusedfor histological and immunohistochemical staining. For human and mousearticularcartilage,Safranin O/Fast green staining is the gold standard for assessing the severity of OA. The severity of articular cartilageOAwasblindlyscoredbytwoexperiencedresearchersusing the Osteoarthritis Research Society International (OARSI) grading system (0–6 points)51,52. To assess the sclerosis of the subchondral boneinmice,wemeasuredtheaveragedistancebetweenfiveevenly distributedmeasuringpointsfromthesubchondralbonemarginto thetopofthetrabecularbone53,54.Synovitis(0fornoinflammation,1 formildinflammation,2formoderateinflammation,3forsevere inflammation)55andosteophyteformation(0fornone,1forprimarily cartilaginous,2formixedcartilaginousandbonywithactivevascular invasion and enchondral ossification, 3 for primarily bony)56 were quantifiedaspreviouslydescribed.

Immunohistochemistry(IHC)andImmunofluorescence(IF)
Representative sections prepared as described above were depar- affinized in xylene, rehydrated through graded ethanol, and then submerged in TE9.0 and incubated overnight at 65°C to expose antigens.ForIHC,sectionsweretreatedwith3%hydrogenperoxide for10min.Sectionswerethenblockedwith1%sheepserumat37°C for 1h, and incubated overnight at 4°C with primary antibodies. Subsequently, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 1h at room tem- perature. Finally, 3, 3-diaminobenzidine was used to observe the chromogen,withhematoxylinoralcianbuleforcounterstaining.For IF,sectionsorcellsseededontoconfocaldisheswereincubatedwith primaryantibodies,thenincubatedinthedarkatroomtemperature for 1h with AlexaFluor488(LifeTechnologies,CatNo.A20000)or AlexaFluor594(LifeTechnologies,CatNo.A10438)conjugatedsec- ondaryantibodies.CellnucleiwerestainedwithDAPI(ThermoFisher Scientific,CatNo.D1306).Imagesofallstainedsectionsandconfocal disheswereacquiredusingaFluoViewFV1000confocalmicroscope (Olympus) and FV10-ASW Viewer (v4.2) software. The ratio of the numberofpositivecellstothetotalnumberofcellswithinmultiple standardizedregionsofinterest(ROIs)wasassessedusingImagePro Plus (v6.0) and Image J (v1.8) software. For mouse knee joints, we analyzedtheratioofthearea/numberofpositivestainingtothetotal area/total number of cells along the joint surface in the femoral condyleandtibialplateauregions.Fortheskin,theratioofthetotal areatothetotallengthoftheepidermallayerwastakenastheaverage thickness of the epidermis. The average intensity of staining was quantifiedastheintegratedopticaldensity(IOD)ofthepositivecell area.Foreachgroup(comprisingatleastthreesamples),atleastthree randomfieldsofviewwereevaluatedfromthreerandomsectionsof eachindividualsample.Detailedinformationregardingtheprimary andsecondaryantibodiesusedforIHCandIFstainingisprovidedin SupplementaryTable2.

HumanOAcartilageexplantculture
Asdescribedpreviously,OAkneecartilagesamplesfrom8patients undergoingtotalkneearthroplastyforadvancedOA(withnohistory ofmedicationtreatment)andcontrolcartilagefrom6patientswith bone tumors undergoing partial knee replacement surgery with no historyofOAwereobtained.AllsamplesweresourcedfromtheThird AffiliatedHospitalofSouthernMedicalUniversity,withapprovalfrom thehospital’sethicscommitteeandwritteninformedconsentfromthe patients. The cartilage specimens were cut into small cubes of 0.5cm^3, washed with sterile PBS, and transferred to 12-well plates containing DMEM/F12 medium (Gibco, Cat No. C11330500BT) sup- plemented with 10% FBS (Gibco, Cat No. 10099141C). Spesolimab (AtaGenix,Cat.No.ATAD00573)wasaddedtotheculturemediumata concentration of 200ng/mL to evaluate its effects on human OA cartilagecells.Thesecartilageexplantswereculturedinmediawithor without spesolimab at 37°C in 5% CO for 14 days, with cartilage 2 explantsfromindividualswithoutOAhistoryservingasblankcontrols. Themediumanddrugwerereplacedeveryotherdayfollowingthe initial steps. After the culture period, cartilage specimens were col- lectedandprocessedaspreviouslydescribedtopreparesectionsfor histologicalandimmunologicalstaining.

Cellculture
Primaryarticularchondrocytes(CCs)wereisolatedfromthefemoral condylesandtibialplateausof3-day-oldC57BL/6Jmice.Inbrief,the chondrocytesweresequentiallydigestedusing0.25%Trypsin-EDTA (Gibco, Cat No. 25200-072) followed by 0.1% collagenase type II (Sigma-Aldrich, Cat No. C-BIOC) at 37°C. The isolated primary chondrocytes were then cultured in DMEM/F12 medium supple- mentedwith10%FBSat37°Cina5%CO atmospheretomaintaina 2 monolayer. Similarly, the C28/I2 cell line (a Human normal chon- drocytecellline;Shzeye,CatNo.ZY-C6492H)wasalsoculturedunder the same conditions of 37°C and 5% CO in DMEM/F12 medium 2 containing10%FBS.
Primarykeratinocytes(KCs)wereisolatedfromtheskinof1–2- day-oldC57BL/6Jmice.Briefly,afterpeelingtheskinfromthemice,it wasrinsedwithDPBS(Gibco,CatNo.14190144)andthentreatedwith 4mg/mL Dispase II (Roche, Cat No. D4693) at 4°C for 18h. Subse- quently,theepidermiswasseparatedfromthedermis,andtheepi- dermiswasincubatedwithTrypLe™Expressenzyme(ThermoFisher, Cat No. 12604013) for 20min to dissociate keratinocytes from the epidermis.Thecellsuspensionwasthenfilteredthrougha70µmmesh sieve and centrifuged to pellet the cells. After removing the super- natant, the isolated primary mouse keratinocytes were cultured at 37°Cin5%CO2 inEpiLife™keratinocytegrowthmedium(Thermo- Fisher,CatNo.MEPI500CA)supplementedwith1%EDGSgrowthfac- tor(ThermoFisher,CatNo.S0125)andCalciumChloride(60µM).The HaCatcellline(aHumanimmortalizedkeratinocytecellline;Procell, CatNo.CL-0090)wasmaintainedat37°Cin5%CO inHaCat-specific 2 medium(Procell,CatNo.CM-0090)containing10%FBS.
Mouse primary synovial fibroblasts (SFs; iCell, Cat No. MIC-iCell- s004)andFLS(Humanfibroblast-likesynoviocytes;iCell,CatNo.iCell- 008a)werebothculturedat37°Cin5%CO intheiCellPrimaryFibro- 2 blastCultureSystemcontaining10%FBS(iCell,CatNo.PriMed-iCell-003).

Cellsstimulation
For senescence induction in keratinocytes, we utilized ultraviolet radiationtoinducesenescenceinwell-conditionedKCsorHaCatcells. When celldensity reached approximately70%,the supernatantwas removed,andathinlayerofDPBSwasadded.Thecellswereirradiated with a medium-wave ultraviolet irradiation device (Analytik Jena; ModelUVM-28)at302nm,deliveringadoseof100mJ/cm^2.Following the irradiation, DPBS was discarded, and complete culture medium wasadded.Thecellswerethenculturedat37°Cina5%CO envir- 2 onmentfor24h.Subsequently,cellularproteinswereharvestedfor the detection of aging-related markers through Western Blot and Immunofluorescenceanalysis.
Forcelltransfection,weutilizedsiRNA(BeijingTsingkeBiotech Co.,Ltd.)tosilenceIL-36RainKCsandIL-36RinCCsandSFs.Following the manufacturer’s instructions, siRNA was diluted in Opti-MEM (ThermoFisher,CatNo.31985070).ThedilutedsiRNA(100nM)was thentransfectedintocellsusingLipofectamine3000(Invitrogen,Cat No.L3000150).After72h,cellularproteinswereextractedtoevaluate theefficiencyoftheknockdown.
FortheapplicationofmIL-36Ra(TargetMol,CatNo.TMPY-04275) or spesolimab (MedChemExpress; Cat No. HY-P99396) to cells, we collectedthesupernatantsfromsenescent/IL-36RaknockdownKCsto induceSFs tobecomeOA-SFs. Then,the supernatantsfromtheco- cultureofsenescent/IL-36RaknockdownKCswithSFswerecollected toinduceCCstobecomeOA-CCs.Onthisbasis,mIL-36Ra(200ng/mL) or spesolimab (0.5μM) was added to culture OA-SFs or OA-CCs to evaluatetheireffectsonOA.Then,wetreatedHaCatcells,FLS,and C28/I2cellsinasimilarmanner.SpesolimabwasaddedtocultureOA- FLSSorOA-C28/I2toevaluatethedrug’seffectonOA.

SynovialfibroblastsMigration
WeconductedascratchassaytoassessthemigrationcapacityofSFs. Well-conditionedSFsorFLSwereseededin6-wellplatesandcultured untiltheyreachednearly100%confluence.Afterremovingtheculture medium,asterile200μlpipettetipwasusedtocreateacross-shaped scratchineachwell.Subsequently,amediumcontaining1%FBSand therespectiveinterventionwasadded.After24h,thecellswerefixed with4%paraformaldehydeandstainedwithammoniumoxalatecrystal violetfor10min.Thestainwasthenwashedoff,andfiverandomfields of view per well were photographed and analyzed to measure the extentofcellmigrationfromtheedgesofthescratch.

Alcianbluestainingofchondrocyteclusters
Well-conditionedCCsorC28/I2cellswereseededinto6-wellplates andcultureduntiltheyreachedapproximately95%confluence.The cells were then digested with trypsin and resuspended in 100μl of DMEM/F12containing10%FBS.Usingapipetteat20μl/dropofthe high-concentrationcellsuspensionwereseededinto12-wellplatesto formtightlyconnectedclustersofchondrocyteswith adiameterof 0.5cm.Afteradheringtotheplatefor6hat37°Cand5%CO2,10% FBS+DMEM/F12medium,alongwiththerespectiveintervention,was added.After24h,thecellswerefixedwith4%paraformaldehydeand stainedwithAlcianBluefor15-20min,followedbywashingoffthe stain.Imageswereacquiredusingascanner,andthestainingaverage intensity was quantified as integrated optical density (IOD) using ImageProPlus(v6.0)softwareforassessment.

SA-β-Galstaining
Toassessthesenescencestatusofcells,weutilizedtheSenescenceβ- GalactosidaseStaining Kit(Beyotime, CatNo.C0602)following the manufacturer’sprotocol.Briefly,theinducedCCsandSFswerefixed for15min.Thefixativewasthenwashedoff,andthecellswereincu- bated with SA-β-Gal staining solution at 37°C in a sealed, light- protectedenvironmentfor16h.Afterwashingoffthestainingsolu- tion, five random areas were photographed in each culture dish to evaluatethedegreeofcellularsenescence.

Westernblotanalysis
Toisolate tissue proteins, the skin, heart, brain,spleen,lungs, kid- neys,andintestinesof3-month-oldand18-month-oldmice,andthe esophagus,heart,brain,spleen,liver,pancreas,lungs,kidneys,sto- machandintestinesofZmpste24fl/fl,Zmpste24-cKO,IL-36Rafl/fl,IL-36Ra- cKO mice were ground in liquid nitrogen. Following experimental induction, proteins from mouse CCs and SFs were extracted for immunoblotanalysis.Briefly,tissuefragmentsorcellswerelysedon iceusing2xSDSlysisbuffer(1MTris-HCl-pH6.8,10%SDS,glycerol, bromophenol blue, and Roche protease inhibitor cocktail). The lysateswerethenseparatedusing10%or12%SDS-polyacrylamidegel electrophoresis, and the separated proteins were transferred to a 0.22μm PVDF membrane (Sigma; Cat No. ISEQ00010) using a wet transfermethod.ThePVDFmembranewasthenblockedwith5%skim milkonarockingplatformatroomtemperaturefor1h,followedby incubationwiththeprimaryantibodyat4°Cfor12-16h.Thenextday, the membrane was incubated with the secondary antibody at roomtemperaturefor1h.Finally,proteinbandswerevisualizedand captured on a chemiluminescence imager (Tanon, Model 5200CE) usingUltraHighSensitivityECLKit(MedChemExpress,CatNo.HY- K1005).Detailed information regardingthe primaryand secondary antibodiesusedforWesternblotanalysisisprovidedinSupplemen- taryTable2.

Quantitativereal-timePCR
Total RNA from induced SFs and FLS was extracted using Trizol reagent (TaKaRa; Cat No. 9108). The concentration was measured usingaNanoDropspectrophotometer(ThermoFisherScientific;Cat No. 840-317500). Following the manufacturer’s instructions, cDNA wassynthesizedfromtotalmRNAusingacDNAsynthesiskit(Vazyme; CatNo.R333-01).RT-PCRwasperformedusingChamQSYBRqPCR MasterMix(Vazyme;CatNo.Q311-02)onaLightCycler®96system (Roche)withLightCycler®96softwareversion1.1.Relativequantifi- cationwasconductedusingthe2-ΔΔCqmethod.Theexpressionof eachgenewasnormalizedtothehousekeepinggeneGAPDH.Primer sequences (Beijing Tsingke Biotech Co., Ltd.) used for RT-PCR are listed in Supplementary Table 3. RT-PCR data were analyzed using Microsoft Excel version 16.36, and graphs were generated using GraphPadPrism9.3.

RNAsequencing
Skintissuefrommice(IL-36Rafl/fl andIL-36Ra-cKO)andchondrocytes from mice (negative control group, senescent keratinocyte identification supernatant-stimulated group) were subjected to RNA sequencing analysis.Inbrief,totalRNAwasextractedusingtrizolreagent(following themanufacturer’sprotocol,withskintissuesinitiallygroundinliquid nitrogen before the addition of trizol), and the quality of RNA was assessedusinganAgilent2100Bioanalyzer(AgilentTechnologies,Palo Alto,CA,USA).mRNAwasthenenrichedandisolatedusingoligo(dT) magneticbeads,followedbytheadditionoffragmentationreagentto fragment the mRNA. Subsequently, first-strand and second-strand cDNA synthesis reactions were prepared (using DNA polymerase I, RNase H,dNTPs,and buffer),and reaction programs were setup to synthesizefirst-strandandsecond-strandcDNA.Thedouble-stranded cDNAwasthenend-repaired,A-tailedatthe3’end,andadapterswere ligatedtothecDNA.PCRamplificationoftheproductfollowed,and PCRproductsweredenaturedintosingle-strandedcircularDNAmole- cules,whichwerethenamplifiedviarollingcircleamplificationtoform DNAnanoballs(DNBs).TheseDNBswereloadedontohigh-densityDNA nanoarraychipsandsequencedatBGIGenomicsCo.,Ltd(Shenzhen, China) using the combinatorial Probe-Anchor Synthesis (cPAS) tech- nology.RawsequencingdatawereprocessedusingSOAPnuke(v1.5.6) to generate clean data. Differentially expressed genes (DEGs) were identified using DESeq2 (v1.34.0), with significance defined as q‑va- lue<0.05calculatedbytheBenjamini–Hochbergmethod.KEGGpath- wayenrichmentanalysiswasperformedusingahypergeometrictest (Phyper)againsttheKEGGdatabase(https://www.kegg.jp/),withq‑va- lue<0.05asthesignificancethresholdforenrichedpathways.GeneSet EnrichmentAnalysis(GSEA)wasperformedusingGSEAv3.0.

LC-MS/MSmassspectrometry-basedproteomics
Wecollecteddorsalskintissuesfrom3-month-oldand18-month-old mice,removedhair,andimmediatelysnap-frozethesamplesinliquid nitrogen. Protein sample preparation and liquid chromatography- tandemmassspectrometry(LC-MS/MS)usinglabel-freequantification (LFQ)wereperformedatPTMBiolabCo.,Ltd.(Hangzhou,Zhejiang, China). Initially, skin tissue proteins were extracted bygrinding the skinofeachgroupintopowderwithliquidnitrogen,followedbythe addition of a fourfold volume of lysis buffer and sonication on ice using a high-intensity ultrasonic processor. After sonication, the supernatantwascollectedbycentrifugation(4°C;12,000×g;10min) toobtainproteinsamples,andtheproteinconcentrationwasdeter- minedusingaBCAProteinAssayKit.Subsequently,theproteinsam- plesunderwent2-3roundsofprecooledacetonewashing,thenwere dissolvedin200mMTEABanddisruptedbysonication.Trypsinwas addedforovernightdigestion.Thesampleswerethenreducedwith 5mM dithiothreitol at 56°C for 30min and alkylated with 11mM iodoacetamide in the dark at room temperature for 15min. The trypsin-digestedpeptidesweredissolvedinsolventAandloadedonto ahomemadereverse-phaseanalyticalcolumn.Peptideseparationwas carriedoutusingamobilephaseofwaterwith0.1%formicacidand2% acetonitrileassolventA,andwaterwith0.1%formicacidand100% acetonitrileassolventB,withagradientof4–22%Bfrom0to70min, 22–30%Bfrom70to84min,30–80%Bfrom84to87min,and80%B from87to90min.Peptideswereionizedusingacapillaryionsource andsubsequentlyanalyzedwithatims-TOFPromassspectrometer. Theionsourcevoltagewassetat2.0kV.Bothprecursorionsandtheir fragmentsweredetectedathighresolutionbytheTOFanalyzer.MS/ MSspectrawereacquiredwithinthemassrangeof100–1700.Data acquisition was performed using the Parallel Accumulation Serial Fragmentation(PASEF)mode.ForeachfullMSscan,10PASEFscans werecollectedforprecursorionswithchargesrangingfrom0to5. Dynamicexclusionwasenabledfor30sectoavoidrepeatedanalysis ofidenticalprecursorions.TheresultingMS/MSdatawereanalyzed usingMaxQuant(version1.6.6.0).Areversedatabasewasincludedto estimatethefalsediscoveryrate(FDR),andacommoncontaminant databasewasusedtoeliminatecontaminationproteinsfromthefinal identification results.Theenzymaticdigestion wassettoTrypsin/P; thenumberofmissedcleavagesiteswassetto2;theminimumpeptide length was set at 7 amino acid residues; the maximum number of modificationsperpeptidewassetto5;theprecursormasstolerance forthefirstsearchandthemainsearchweresetto20ppmand20 ppm,respectively,andthemasstoleranceforsecondaryfragmentions wassetto20ppm.Carbamidomethyl(C)wassetasafixedmodifica- tion for cysteine alkylation, with variable modifications including methionine oxidation, protein N-terminal acetylation, and deamida- tion (NQ). The FDR forprotein identificationand peptide-spectrum matches(PSMs)wassetto1%.
Weidentifiedsignificantlydifferentiallyabundantproteinsusinga combinationoffoldchange(FC>1.5or<1/1.5)andadjustedp-values. Specifically,wecalculatedthefoldchange(FC)as:FC =Mean(R ,i A/Bk ik ∈A)/Mean(R ,i∈B),whereRrepresentstherelativeproteinquanti- ik ficationvalue,irepresentsthesample,andkrepresentstheprotein.To assess statistical significance, p-values were calculated using an unpairedtwo-tailedStudent’sttestonlog2-transformedvalues:P =T. k test(Log2(R ,i∈A),Log2(R ,i∈B)).Tocorrectformultipletesting,the ik ik Benjamini-Hochbergprocedurewasappliedtocontrolthefalsedis- covery rate (FDR), and q-values were obtained. Proteins with an adjustedq-value<0.05andanabsolutefoldchange>1.5(i.e.,FC>1.5 or FC<1/1.5) were considered significantly differentially expressed, ensuringbothstatisticalandbiologicalrelevance.

Elisaassays
Serum was collected from both young and aged mice, young and elderlyhumans,andsynovialfluidfromelderlyOApatients.Wealso collectedserumsamplesfromD-Galactose-treatedandtheircontrol male mice. Additionally, we collected serum from Zmpste24fl/fl, IL- 36Rafl/fl, Zmpste24-cKO, and IL-36Ra-cKO male mice, as well as from Zmpste24-cKO, IL-36Ra-cKO male mice and their control male mice aftertheapplicationofMN-mIL-36RaorMN-SPE.Supernatantsfrom UV-inducedsenescentKCsandHaCatcellswerealsocollected.The levelsofIL-36α,IL-36β,IL-36γ,IL-36RaandD-Galinthesesampleswere determinedusingELISAkitsforIL-36α(Cat.No.:MM-45615M2),IL-36β (Cat.No.:MM-45619M2),IL-36γ(Cat.No.:MM-45212M2),IL-36Ra(Cat. No.: MM-48043M2), Spesolimab (Cat. No.: MM-95430O1) and D-Galactose(Cat.No.:MM-45619M2)accordingtothemanufacturer’s instructions(JiangsuMeimianIndustrialCo.,Ltd).

D-Galactosecontentassay
TheD-galactosecontentinserumsamplesfromD-Galactose-induced prematurelyagedskinmalemice(8weeks)andtheircontrolC57BL/6J malemicewasmeasuredusingaD-GalactoseContentAssayKit(ADS- W-TDX046, Jiangsu Meimian Industrial Co., Ltd) according to the manufacturer’sinstructions.Briefly,Serumsampleswerecentrifuged at12,900×gfor10minatroomtemperature,andthesupernatantwas collected. The assay uses D-galactose reacting with galactose dehy- drogenasetoformNADH,whichproducesayellowcompoundwithan absorbance peak at 450nm. Supernatants were added to a 96-well plate with the kitreagents, incubated at25°C, and absorbancewas measuredat450nm(A1).Afteraddingasecondreagent,theplatewas incubatedfor20min,andabsorbance(A2)wasmeasuredagain.The change in absorbance (ΔA=A2 - A1) was used to calculate the D-galactosecontent,expressedasμg/ml.

Statisticalanalysis
Allreporteddatawerederivedfromatleastthreeindependentbio- logicalreplicates,withthenumberofbiologicalreplicatesindicatedby “n”inthefigures.Alldataoriginatefromthesebiologicalreplicatesand are presented as mean±standard deviation (S.D.). We used the Shapiro-Wilk testto assessdata normality. The unpaired,two-tailed Student’s t test or Mann-Whitney rank sum test (for non-normally distributeddata)wasusedtocomparetwoindependentgroups.For comparing multiple values, we used a one-way analysis of variance (ANOVA) with Tukey’s post hoc testor Kruskal-Wallistest(fornon- normallydistributeddata)withDunn’smultiplecomparisonstest.All statistical tests used were two-sided. Values of p<0.05 were con- sideredstatisticallysignificant.Finally,wegeneratedthegraphsusing GraphPadPrismversion9.3.Fortheconstructionofallanimalandcell models,aswellasdrugadministration,theoperatorswereblindedto thegroupinformation.Weensuredblindingduringthedatacollection processbyassigningrandomIDstoallsamples,preventingaccessto groupinformation.Histologicalstaining,WB,RT-PCR,IHC,andIFwere carriedoutbyindividualsotherthantheexperimentdesigners.For histological analysis, the analysts were blinded to the sample infor- mation,includingthegenotypeofthemice,anydrugtreatments,or surgicalconditions.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper the authors present a paper focuses on a very interesting area, namely the treatment of osteoarthritis. There is currently a shortage of therapies to slow the progression of this disease. Likewise, the study of the connection between the skin and the joint is also very innovative and opens the door to other types of treatments.\nAuthors suggest that IL-36 agonists are age-related systemic inflammatory factors released from skin to joints and contribute to OA development. IL-36Ra shows potential clinical translational value for OA treatment, and targeting IL-36R signaling in aged skin with microneedles represents a promising disease-modifying approach.\nThe manuscript is well written, clear and easy to read. The authors have performed a wide variety of in vitro and in vivo models to prove their hypothesis. This methodology is well described and is easy to understand.\nCOMMENTS:\n1. Figure 1 d: pictures showing the osteophytes are not clear. How the ssubchondral bone was evaluated? My impression is that the principal modification of SBP is in sclerosis, however I don’t see clear changes in the thickness and synovium.\n2. Figure 1j: OARSI score and articular cartilage of control and control serum are very similar.\n3. Figure 2 o, shows the correlation between the levels of cytokines in serum and synovial fluid. However, the concentration of these cytokines are not showed in the figure and are not described in the text. Please could you give these data. This information is very important to know the real origin of the cytokine synthesis.\n4. Figure 4h shows results at 10 months only in sham model. This figure does not shows results of OA model, why?\n5. Figure 4i. there are not differences in the subchondral bone and osteophytes, comment in the text is not right. Reduction of proteoglycans is not quantified.\n6. Figure 5i and 5j: Pictures don´t correlate to much with the data show in the graphics. From my point of view there is not to much differences on the OARSI score between the different models. This comment is similar to the Figure 5l and 5m.\n7. Figure 7. Spesolimab shows potential therapeutic efficiency for human OA and attenuates experimental OA in mice via microneedle delivery. Results showed in the panels l,m, o and p are very light.\n8. Refences 24 and 28 are duplicated.\n9. Overall, histological results (pictures) of the OA animal model are unclear and unconvincing. Differences between groups are often very weak, mainly OARSI score, subchondral bone, osteophytes and even synovial. From my point of view this is the most important weakness of the manuscript. The hypothesis is very interesting, and the data shown in the skin are clearer than those shown at the joint level. The authors should confirm their results in a clearer and more convincing animal model of osteoarthritis. The data also do not clearly demonstrate the connection between skin and joints. Knowing the levels of cytokines in serum and synovial fluid would help to better understand this connection.", "reviewer_2": "The article, \"Aged skin exacerbates experimental osteoarthritis via enhanced IL-36R signaling,\" provides valuable insights into the relationship between aging skin and the IL-36 signaling pathway in the context of osteoarthritis (OA). The results presented are both novel and compelling, particularly the discovery that aged skin contributes to IL-36 agonist signaling, which in turn exacerbates OA progression. The study effectively supports this hypothesis through a series of well-designed experiments, providing strong evidence for the potential therapeutic benefits of blocking IL-36 signaling to improve OA outcomes. The overall flow of the study is coherent, and the experimental design aligns well with the research objectives.\nHowever, there are a few areas that could be improved to further strengthen the manuscript. Addressing these points would enhance the clarity and impact of the findings, ultimately resulting in an even more robust contribution to the field.\n1. The scale bars in the images for articular cartilage, subchondral bone, synovium, and osteophytes appear to be inconsistent. Specifically, if the scale bar in the subchondral bone plate (SBP) image represents 50 µm, then the bar graphs measuring SBP thickness may be inaccurate. This issue seems to persist across multiple figures. It would be beneficial to double-check the magnification levels and scale bars for all images to ensure proper calibration and accuracy.\n2. It is important to clearly indicate which colors correspond to specific markers in the fluorescent staining images. For example, as noted in Figure 5, mentioning that green and red represent different markers would help readers follow the content more easily. This should be consistent across all relevant figures.\n3. The explanations in the manuscript would benefit from more detailed descriptions of the markers and cells studied. For example, it would be helpful to clarify what the specific markers represent and the significance of their increase or decrease in the context of the study.\n4. The timelines, depicted with arrows to represent weekly or biweekly intervals, appear to be drawn in an unclear or inconsistent manner.\n5. There is an inconsistency between the text and figure legend regarding the administration of AAV2-Cre or D-gal. While the text mentions topical application, the figure indicates intradermal injection, and the mouse scheme seems to suggest liquid application on top of the skin. It is important to match these details to avoid confusion.\n6. In Figure 4c, while SBP thickness appears to be increased in the IL-36Ra fl/fl mouse, this was not statistically significant. However, the text suggests that the knockout (KO) mouse showed significant improvement in all measured factors, which is not consistent with the data.\n7. Certain abbreviations, such as SPE and SNL, are used without prior explanation.\n8. In Supplementary Figure 8c, the IL-36R-KD SNL group did not show recovery, but the main text could be misinterpreted to suggest that recovery occurred.\n9. The concluding statement in Figure 5, “Thus, these data suggest that loss of IL-36Ra in keratinocytes leads to increased IL-36 agonist secretion and induces chondrocyte catabolism and synovial inflammation by activating pro-inflammatory signaling, including NF-κB/JAK-STAT/MAPK pathways,” could benefit from a more detailed summary of the results. The explanation currently feels somewhat weak and should more clearly highlight how the results lead to this conclusion.\n10. In Figures 6 and 7, the comparison of OA recovery through microneedles (MN) should include both control groups such as sham-treated mice and intra-articular (i.a.) injection treatment groups for a more comprehensive comparison.", "reviewer_3": "The authors present an interesting manuscript on skin aging and bone health, mediated by skin derived, circulating levels of IL-36 receptor agonists that seem to have a systemic effect. This is a fascinating new aspect of skin aging having a systemic effect.\nMajor\nAs far as aspects related to proteomics and transcriptomics are concerned, did the authors use adjusted p values for multiple testing? Which correction for multiple testing correction did they use for proteomics and transcriptomics?\nMinor\nIn the introduction, the authors might want to explain a bit more why they think their specific model is an appropriate model reflecting human skin aging."}, "author_response": "Point-to-point responses to the reviewers’ comments\nDear editor and reviewers,\nThank you very much for your recommendation to revise our manuscript. To address the comments,\nwe have made further revisions to our manuscript. Our point-by-point responses to these comments\nare provided below. The authors sincerely hope that our replies have adequately addressed your\nconcerns.\nReviewer #1 (Remarks to the Author):\nIn this paper the authors present a paper focuses on a very interesting area, namely the\ntreatment of osteoarthritis. There is currently a shortage of therapies to slow the progression of\nthis disease. Likewise, the study of the connection between the skin and the joint is also very\ninnovative and opens the door to other types of treatments.\nAuthors suggest that IL-36 agonists are age-related systemic inflammatory factors released\nfrom skin to joints and contribute to OA development. IL-36Ra shows potential clinical\ntranslational value for OA treatment, and targeting IL-36R signaling in aged skin with\nmicroneedles represents a promising disease-modifying approach.\nThe manuscript is well written, clear and easy to read. The authors have performed a wide\nvariety of in vitro and in vivo models to prove their hypothesis. This methodology is well\ndescribed and is easy to understand.\nCOMMENTS:\n1. Figure 1 d: pictures showing the osteophytes are not clear. How the subchondral bone was\nevaluated? My impression is that the principal modification of SBP is in sclerosis, however I don’\nt see clear changes in the thickness and synovium.\nResponse: We greatly appreciate your valuable feedback on our study. We have carefully\nexamined all osteophyte and synovial images, including Fig. 1d. We have re-photographed and\nupdated the figures of osteophytes and synovium in Fig. 1d, Fig. 1j, Fig. 4b, Fig. 4h, Fig. 6i,\nFig. 6l, Fig. 7l, Fig. 7o, Supplementary Fig. 2b, Supplementary Fig. 9c, and Supplementary\nFig. 10c to better present representative images.\nAs you mentioned, subchondral bone sclerosis is indeed an important feature of OA\nprogression, typically including increased bone density, thickening of the subchondral bone\nplate, and osteophyte formation. Regarding the thickness of the subchondral bone plate in mice,\nwe assessed it as follows: we selected three sagittal slices spaced approximately 30μm apart\nfrom the load-bearing area of the mouse knee joint. Using Safranin O-fast green staining, we\nwere able to clearly distinguish the boundary between cartilage and bone tissue, allowing for\naccurate measurement of subchondral bone thickness. The method we used for measuring\nsubchondral bone thickness is based on a well-established approach reported in the literature1,\n2. Specifically, we selected five evenly distributed measurement points between the cartilage\nsurface and the top of the trabecular bone, calculated the average as the representative value for\neach slice, and used the mean of the three slices as the final representative value for subchondral\nbone thickness. This method reflects the overall changes in subchondral bone and provides an\nobjective basis for evaluating subchondral bone sclerosis.\n2. Figure 1j: OARSI score and articular cartilage of control and control serum are very similar.\nResponse: We greatly appreciate your professional comments. In Fig. i-l, we used the DMM\ntechnique to establish a mouse OA model and injected small amounts of serum from young\nmice (Control serum) or serum from D-Gal-induced skin aging mice (D-Gal serum) into the\njoint cavity weekly for eight weeks. The OARSI scores indicated that the D-Gal serum group\nexhibited more severe OA progression compared to the model-only group (Control), suggesting\nthat aged D-Gal serum may contain pro-inflammatory factors or metabolic products that\naccelerate the pathological process of OA.\nHowever, the progression of OA in the Control serum group did not differ significantly from\nthe Control group, suggesting that the serum from young mice contains relatively low levels of\ninflammatory factors and is free of proinflammatory factors commonly observed in models of\nageing or OA. While the serum contains some anti-inflammatory and repair factors, these may\nnot be sufficient to significantly influence the progression of OA. Furthermore, the\ninflammatory factors in young serum may be inadequate to counteract the pathological response\ninduced by the mechanical damage of the DMM surgery. Therefore, the effect of young mouse\nserum on OA progression is limited and did not significantly affect the OA process in the short\nterm, and the OARSI score and articular cartilage between the Control and Control serum\ngroups are similar.\n3. Figure 2 o, shows the correlation between the levels of cytokines in serum and synovial fluid.\nHowever, the concentration of these cytokines are not showed in the figure and are not\ndescribed in the text. Please could you give these data. This information is very important to\nknow the real origin of the cytokine synthesis.\nResponse: Thanks for your constructive comments. Regarding the detailed data and\nconcentrations of IL-36α, β, and γ in human serum and synovial fluid, we have already provided\nthe detailed data in the Raw Data, as shown below:\nIL-36α (pg/mL) IL-36β (pg/mL) IL-36γ (pg/mL)\nSerum Synovium Serum Synovium Serum Synovium\nfluid fluid fluid\n306.2154 341.2247 282.2238 338.7454 439.2212 530.8976\n276.8529 303.9568 372.197 376.811 434.3961 524.4642\n324.2847 346.8713 319.1359 347.9734 471.3883 537.331\n292.6635 332.19 344.5129 367.583 350.7615 487.4719\n319.7674 370.5872 307.6008 347.9734 418.3125 513.2057\n268.9476 297.1808 327.2104 335.2849 464.9549 521.2475\n328.802 360.4232 354.8944 364.1224 439.2212 527.6809\n309.6034 314.1207 361.8154 401.0345 476.2134 546.9812\n334.4487 376.2339 294.9123 347.9734 452.088 518.0307\nIn order to ensure that readers can clearly and intuitively understand the relevant data, the\nconcentration ranges for IL-36α, β, and γ have been explicitly mentioned in the text. The revised\ntext is shown below, with the added content highlighted in yellow:\n(line 156) We also found positive correlations of elevated expression of IL-36 agonists\nbetween serum and synovial fluid in aged OA patients, with IL-36α concentrations 301.70 ±\n32.75 pg/mL in serum and 319.76 ± 27.11 pg/mL in synovial fluid, IL-36β concentrations\n319.14 ± 25.18 pg/mL in serum and 335.28 ± 32.56 pg/mL in synovial fluid, IL-36γ\nconcentrations 418.31 ± 20.92 pg/mL in serum and 513.21 ±33.77 pg/mL in synovial fluid. The\ndetermination coefficients (R²) of IL-36α, β, and γ were 0.7943, 0.6233, and 0.8088,\nrespectively.\n4. Figure 4h shows results at 10 months only in sham model. This figure does not shows results\nof OA model, why?\nResponse: Thanks for your comments. In our experimental design, we did indeed include 10-\nmonth-old IL-36Ra fl/fl and IL-36Ra cKO mice to represent the physiological state of middle-\naged to elderly stages with the label “sham-10M.” However, we did not include a DMM-\ninduced OA group in the 10-month-old mice (i.e., no “OA-10M” group) for the following\nreasons:\nFirst, in Fig. 4g-j, our main research objective was to investigate the effects of IL-36Ra fl/fl\nand IL-36Ra cKO mice on joint health during natural aging, rather than in a DMM-induced OA\nmodel. Second, in Fig. 4a-e, we have already compared and analyzed the differences between\nIL-36Ra fl/fl and IL-36Ra cKO mice in the DMM-induced OA model. Finally, performing DMM\nsurgery on the 10-month-old mice could introduce additional variables, such as age-related\ndeclines in healing capacity and bone remodeling changes, which may affect the comparison of\njoint status in IL-36Ra fl/fl and IL-36Ra cKO mice. Therefore, we chose to perform DMM\nmodeling in young mice and only observed the natural joint status in the 10-month-old group.\nWe have corrected “sham-10M” to 10M in Fig. 4h to avoid potential misunderstanding. Thank\nyou again for your attention to the experimental logic.\n5. Figure 4i. there are not differences in the subchondral bone and osteophytes, comment in the\ntext is not right. Reduction of proteoglycans is not quantified.\nResponse: Thank you for your thorough review of our study. We sincerely apologize for the\ninconsistency in the original manuscript regarding the description of subchondral bone\nthickness and osteophyte formation quantification. We have removed the relevant descriptions\nfrom the manuscript to ensure that the text is consistent with the data.\nRegarding the quantification of proteoglycans in articular cartilage, as you pointed out, the\noriginal manuscript did not provide a clear quantitative description. However, we have assessed\nthe pathological changes in cartilage using Safranin O-fast green staining and OARSI scoring,\nwhich can reflect the accumulation of proteoglycans in cartilage and their pathological changes.\nThese methods are important indicators for evaluating OA progression. While proteoglycan\nquantification has its value in some studies, we believe that using OARSI scoring as the primary\nanalysis method aligns better with the overall research objectives and simplifies data\npresentation, making the results more prominent.\nTo avoid unnecessary repetition and redundancy, we have removed the related descriptions\nof proteoglycan quantification from the manuscript. The revised manuscript now reads:\n(lines 213-215) IL-36Ra-cKO mice exhibited increased cartilage destruction and\naggravated synovitis in the joints (Fig. 4h–i).\n6. Figure 5i and 5j: Pictures don´t correlate to much with the data show in the graphics. From\nmy point of view there is not too much differences on the OARSI score between the different\nmodels. This comment is similar to the Figure 5l and 5m.\nResponse: Thank you very much for your professional and comprehensive comments.\nRegarding the relationship between the data and images in Fig. 5i, 5j, 5l, and 5m (which are\nactually Fig. 6i, 6j, 6l, and 6m), we understand your concern and have carefully reviewed the\ndata. Our study aims to evaluate the therapeutic effects of MN-mIL-36Ra (microneedles loaded\nwith mIL-36Ra) on experimental OA in mice. Therefore, we established DMM-induced OA in\nZmpste24 fl/fl, Zmpste24 cKO, IL-36Ra fl/fl, and IL-36Ra cKO mice and applied MN-mIL-36Ra\nto the mice’s skin. Samples were collected at week 8 post-surgery. The experimental results\nshowed that the application of MN-mIL-36Ra significantly improved the OA symptoms in\nZmpste24 fl/fl, Zmpste24 cKO, IL-36Ra fl/fl, and IL-36Ra cKO mice.\nWe noticed that the images in the Zmpste24 cKO and IL-36Ra cKO mouse groups with\nMN-mIL-36Ra treatment were not strongly representative, we sincerely apologize for this\noversight. Therefore, we re-photographed the high-magnification images of cartilage in Fig. 6i\nand 6l to show the most significant differences in the sections of the cartilage surface, so that\nthe pictures are closely related to the data shown in the graphics.\n7. Figure 7. Spesolimab shows potential therapeutic efficiency for human OA and attenuates\nexperimental OA in mice via microneedle delivery. Results showed in the panels l, m, o and p\nare very light.\nResponse: Thank you for this your comment. We sincerely appreciate your recognition of the\npotential therapeutic effects of spesolimab administered via microneedles in both human and\nmouse OA. However, we also observed that spesolimab delivered through microneedles did not\nshow sufficiently significant improvement in subchondral bone sclerosis and osteophyte\nformation in OA mice. We believe this could be related to the stage of disease progression and\nthe characteristics of the DMM model. During OA progression, the evaluation of subchondral\nbone sclerosis and osteophyte formation often does not show statistically significant differences,\nwhich is consistent with the characteristics of the early to mid-stage OA. The DMM model\n(medial meniscal destabilization) we used generally shows milder subchondral bone changes\nin the early to mid-stage compared to the ACLT model (anterior cruciate ligament transection),\nwhich typically exhibits more pronounced subchondral bone sclerosis and osteophyte\nformation3, 4. This explains why changes in subchondral bone thickness and osteophyte\nformation did not reach statistical significance in some groups in our study.\nWe have also re-photographed and updated the high-magnification images of cartilage in\nFigure 7l, m, o and p with more representative images. Although MN-SPE did not achieve\ncomprehensive therapeutic effects on experimental OA in mice, significant improvements were\nstill observed in OARSI scores, synovitis scores, and Von Frey testing. These results\ncollectively demonstrate the potential efficacy of spesolimab in OA treatment to some extent.\n8. Refences 24 and 28 are duplicated.\nResponse: We sincerely apologize for this mistake. We have corrected the citation and ensure\nthe uniqueness of each reference number, as well as update the reference list accordingly.\n9. Overall, histological results (pictures) of the OA animal model are unclear and unconvincing.\nDifferences between groups are often very weak, mainly OARSI score, subchondral bone,\nosteophytes and even synovial. From my point of view this is the most important weakness of\nthe manuscript. The hypothesis is very interesting, and the data shown in the skin are clearer\nthan those shown at the joint level. The authors should confirm their results in a clearer and\nmore convincing animal model of osteoarthritis. The data also do not clearly demonstrate the\nconnection between skin and joints. Knowing the levels of cytokines in serum and synovial\nfluid would help to better understand this connection.\nResponse: Thank you very much for acknowledging our study. We greatly appreciate your\nprofessional and comprehensive comments, your feedback is crucial for improving the quality\nof the research, especially concerning the significance of histological data and the subtle\ndifferences observed in the OA animal model. We have thoroughly reviewed the minor\ndifferences in histological results, inter-group OARSI scores, subchondral bone thickness,\nosteophytes, and synovial scores in the OA animal model. We understand that these differences\nmay raise concerns about the significance of the results. Therefore, to present the experimental\nresults more effectively and ensure the credibility of our conclusions, we have appropriately\nrenewed some histological images from the OA animal model to ensure they clearly and\naccurately support our conclusions. Additionally, we have included detailed data on cytokine\nlevels in serum and synovial fluid and have added relevant descriptions in the revised\nmanuscript.\nOnce again, thank you for your meticulous review and constructive feedback. We sincerely\nhope that our revisions address your concerns and further enhance the quality of our research.\nReviewer #2 (Remarks to the Author):\nThe article, \"Aged skin exacerbates experimental osteoarthritis via enhanced IL-36R\nsignaling,\" provides valuable insights into the relationship between aging skin and the IL-36\nsignaling pathway in the context of osteoarthritis (OA). The results presented are both novel\nand compelling, particularly the discovery that aged skin contributes to IL-36 agonist signaling,\nwhich in turn exacerbates OA progression. The study effectively supports this hypothesis\nthrough a series of well-designed experiments, providing strong evidence for the potential\ntherapeutic benefits of blocking IL-36 signaling to improve OA outcomes. The overall flow of\nthe study is coherent, and the experimental design aligns well with the research objectives.\nHowever, there are a few areas that could be improved to further strengthen the manuscript.\nAddressing these points would enhance the clarity and impact of the findings, ultimately\nresulting in an even more robust contribution to the field.\n1. The scale bars in the images for articular cartilage, subchondral bone, synovium, and\nosteophytes appear to be inconsistent. Specifically, if the scale bar in the subchondral bone plate\n(SBP) image represents 50 µm, then the bar graphs measuring SBP thickness may be inaccurate.\nThis issue seems to persist across multiple figures. It would be beneficial to double-check the\nmagnification levels and scale bars for all images to ensure proper calibration and accuracy.\nResponse: Thank you for pointing out the inconsistency in the scale bars of the images. We\nhave carefully checked the scale bars and magnification of all images and indeed found that\nsome images had inaccurate scale bar labels. We have corrected the relevant figure legends and\nensured that the scale bars and magnifications are consistent across all images to guarantee the\naccuracy and comparability of the data.\n2. It is important to clearly indicate which colors correspond to specific markers in the\nfluorescent staining images. For example, as noted in Figure 5, mentioning that green and red\nrepresent different markers would help readers follow the content more easily. This should be\nconsistent across all relevant figures.\nResponse: We fully agree with your suggestion to use text labels consistent with the colors of\nthe immunofluorescence staining in the images, as this will help readers more easily understand\nthe content. Therefore, we have reviewed and revised the text labels for all immunofluorescence\nimages in the figures. For all images with dual immunofluorescence co-localization, we have\nused text labels in colors corresponding to the fluorescence. For images with single\nfluorescence staining labeled in black text, we have changed all the black text to match the\nfluorescence colors. The detailed corrections are as follows:\n Fig. 1b: p16 and Lamin B1\n Fig. 1f: Col2a1 and PDPN\n Fig. 2b: Lamin B1\n Fig. 2i: p16 and Lamin B1\n Fig. 3j and 3l: p-p38, p-JNK, p-ERK, p-p65, p-STAT3\n Fig. 4d: Col2a1, PDPN, and p16\n Fig. 7b: Col2a1\n Fig. 7c: Col2a1 and Aggrecan\n Supplementary Fig. 1b and 1d: Zmpste24\n Supplementary Fig. 1e: p16 and LaminA/C\n Supplementary Fig. 2e: Col2a1 and PDPN\n Supplementary Fig. 5c and 5d: IL-36Ra\n Supplementary Fig. 6a: IL-36Ra\n Supplementary Fig. 6d: Col2a1, PDPN, and p16\n Supplementary Fig. 7c and 7d: CD31 and PDPN\n Supplementary Fig. 8d: p-p38, p-JNK, p-ERK, p-p65, p-STAT3\n Supplementary Fig. 9e and 10e: Col2a1 and PDPN\n Supplementary Fig. 11f and 11i: Col2a1 and PDPN\n Supplementary Fig. 12i and 12l: Col2a1 and PDPN\n3. The explanations in the manuscript would benefit from more detailed descriptions of the\nmarkers and cells studied. For example, it would be helpful to clarify what the specific markers\nrepresent and the significance of their increase or decrease in the context of the study.\nResponse: Thanks for your insightful and constructive comments. Indeed, further clarification\nof the descriptions of markers and cells will help readers better understand the background and\nresults of the study. In the Methods and Materials section, we have provided a detailed\nintroduction to the HaCat, FLS, and C28/I2 cell lines. For specific markers, the modifications\nin the original text are as follows (the added text is marked in yellow):\n1. (lines 78-80) Premature skin aging was successfully induced in D-gal-treated mice, which\ndisplayed a thinner epidermis, decreased cell numbers, and immunofluorescence staining\nrevealed an increased number of p16-positive keratinocytes and a decreased Lamin B1\nexpression, both of which are markers of cellular senescence (Fig. 1b–c).\n2. (lines 84-86) Immunostaining showed increased expression of the catabolic marker\nmetalloproteinase 13 (MMP13) and the fibrosis and lymphatic angiogenesis marker\npodoplanin (PDPN), along with decreased expression of the anabolic marker Collagen type\nII (Col2a1) in D-gal-treated mice, while the opposite was observed in skin-removed mice\n(Fig. 1f–g).\n3. (lines 225-226) In the synovium, we observed gradual increases in IL-36R positive cells\nwith age. and using specific markers, we confirmed that the majority of IL-36R positive\ncells were synovial fibroblasts, as indicated by PDPN expression, while no significant co-\nlocalization was observed with F4/80-positive macrophages (Fig. 5a).\n4. (lines 101-105) We confirmed high deletion efficiency of Zmpste24 in epidermal\nkeratinocytes but not in other organs, and compared to control mice, the skin of Z24-cKO\nmice showed a thinner epidermis, decreased cell numbers, loss of rete ridges, and\nremarkably increased senescent keratinocytes, manifested as an increase in p16 and a\ndecrease in Lamin A/C expression, a nuclear envelope stability protein whose processing\nis impaired due to Zmpste24 knockout (Supplementary Fig. 1 b–f).\n4. The timelines, depicted with arrows to represent weekly or biweekly intervals, appear to be\ndrawn in an unclear or inconsistent manner.\nResponse: We apologize for the unclear information. We have carefully reviewed and redrawn\nthe relevant diagrams, including Fig. 1a, Fig. 1i, Fig. 4a, Fig. 6e, Fig. 7k, Supplementary Fig.\n2a, Supplementary Fig. 9a and Supplementary Fig. 10a, to ensure that the arrows in the\ndiagrams correspond accurately with the intervention time. We sincerely appreciate your\ncareful review.\n5.There is an inconsistency between the text and figure legend regarding the administration of\nAAV2-Cre or D-gal. While the text mentions topical application, the figure indicates\nintradermal injection, and the mouse scheme seems to suggest liquid application on top of the\nskin. It is important to match these details to avoid confusion.\nResponse: Thanks for your constructive comments. We have noted your concerns, and in\nresponse, we have re-examined the descriptions of the use of AAV-Cre and D-gal in the original\ntext, figure legends, and images. Our descriptions indeed contained some confusion. In the\nMethods and Materials section, we have provided detailed descriptions of the usage of D-gal\nand AAV-Cre (D-gal was applied topically to the skin; AAV-Cre was applied topically to the\nskin and assisted by a dermaroller to facilitate AAV-Cre entry into the epidermis). To avoid any\nfurther concerns from the readers, we have made the following revisions: we have added a\ndropper for skin application in Fig. 1a, and included the dermaroller in Fig. 4a, Fig. 6e, Fig.\n7k, Supplementary Fig. 2a, Supplementary Fig. 9a and Supplementary Fig. 10a. Thank\nyou again for your thorough review.\n6. In Figure 4c, while SBP thickness appears to be increased in the IL-36Ra fl/fl mouse, this\nwas not statistically significant. However, the text suggests that the knockout (KO) mouse\nshowed significant improvement in all measured factors, which is not consistent with the data.\nResponse: Thank you for pointing out the inconsistency contents. We indeed observed that in\nthe data, compared to IL-36Ra cKO mice, the SBP thickness of IL-36Ra fl/fl mice appeared to\nbe slightly increased overall. However, we mistakenly stated in the text that IL-36Ra cKO mice\nexhibited more severe subchondral bone sclerosis than IL-36Ra fl/fl mice, which is inconsistent\nwith the data. Therefore, we have corrected the description in the text to accurately reflect the\ndata results. Corrected content:\n(lines 202-205) Importantly, at 2 months after DMM surgery, safranin O staining revealed that\nIL-36Ra-cKO mice displayed greater cartilage degradation, synovitis, osteophyte maturity, and\nmarkedly increased OARSI scores than IL-36Ra fl/fl mice. However, no significant differences\nin SBP thickness were observed between IL-36Ra cKO and IL-36Ra fl/fl mice (Fig. 4b–c).\nThank you again for your meticulous approach to our research, which has further improved\nthe accuracy of the article.\n7. Certain abbreviations, such as SPE and SNL, are used without prior explanation.\nResponse: We sincerely appreciate your careful review. To ensure that readers can smoothly\nunderstand the content of the article, we have added explanations for these abbreviations at the\ncorresponding locations where they first appear in the manuscript. The modified content is\nmarked in yellow, as follows:\n1. (lines 144-148) Immunostaining and western blot analysis confirmed successfully induced\nsenescence, which was accompanied by decreased IL-36Ra in cultured keratinocytes\n(Supplementary Fig. 4a–c), and increased IL-36 agonists in the supernatant liquor (SNL)\nvia ELISA analysis (Supplementary Fig. 4d).\n2. (lines 233-236) Subsequently, we cultured mouse keratinocytes and induced senescence with\nUVB irradiation. We next investigated the effects of supernatant liquor from aging\nkeratinocytes (Aging SNL) on chondrocytes or synovial fibroblasts with mIL-36Ra or IL-36R\nknockdown (Fig. 5c).\n3. (lines 254-257) Interestingly, we observed similar changes, with increased inflammation in\nsynovial fibroblasts and chondrocyte catabolism with the stimulation of supernatant liquor\nfrom IL-36Ra knockdown keratinocytes (IL-36Ra-KD SNL), both of which were recovered\nby mIL-36Ra and IL-36R knockdown (Fig. 5i–l, Supplementary Fig. 8c).\n4. (lines 344-346) Next, we established DMM-induced OA in control, Z24-cKO, or IL-36Ra\ncKO mice, and covered the skin of mice with MN-SPE (microneedles loaded with spesolimab)\nto examine its effect on OA protection (Fig. 7k).\n8. In Supplementary Figure 8c, the IL-36R-KD SNL group did not show recovery, but the main\ntext could be misinterpreted to suggest that recovery occurred.\nResponse: We apologize once again for our mistake. In the cultured mouse keratinocytes, we\nused small interfering RNA to knockdown IL-36Ra and stimulated synovial fibroblasts and\nchondrocytes with the resulting supernatant. We observed that under stimulation by IL-36Ra\nknockdown keratinocyte supernatant (IL-36Ra-KD SNL), inflammation in synovial fibroblasts\nand catabolism in chondrocytes increased. Both of these effects were restored by knocking\ndown mIL-36Ra and IL-36R (Fig. 5h–l). Similarly, under stimulation with IL-36Ra-KD SNL,\nupregulation of p-p38, p-ERK1/2, p-JNK, p-p65, and p-STAT3 was observed in synovial\nfibroblasts and chondrocytes, and this was reversed upon adding mIL-36Ra (Supplementary\nFig. 8c).\nWe realized that in Supplementary Fig. 8c, we mistakenly referred to \"IL-36Ra-KD\nSNL\" as \"IL-36R-KD SNL,\" which led to confusion between IL-36Ra and IL-36R. To resolve\nthis, we have corrected the text in the image to accurately reflect the data.\n9. The concluding statement in Figure 5, “Thus, these data suggest that loss of IL-36Ra in\nkeratinocytes leads to increased IL-36 agonist secretion and induces chondrocyte catabolism\nand synovial inflammation by activating pro-inflammatory signaling, including NF-κB/JAK-\nSTAT/MAPK pathways,” could benefit from a more detailed summary of the results. The\nexplanation currently feels somewhat weak and should more clearly highlight how the results\nlead to this conclusion.\nResponse: Thank you for your valuable comments. Based on your suggestion, we have\nprovided a more detailed summary and explanation of the concluding statements about Figure\n5 in revised manuscript. We have strengthened the connection between the experimental results\nand the conclusions, to ensure that the significances of the results are presented more clearly.\nThe revised contents are shown as below:\n(lines 240-243) Furthermore, in cultured mouse primary chondrocytes, loss of proteoglycan\ncontent, increased cellular senescence, inhibited anabolism, and enhanced catabolism were\nnoticed upon stimulation with Aging SNL, all of which were recovered by mIL-36Ra and IL-\n36R knockdown (Fig. 5f–g). This suggests that the Aging SNL induces catabolic processes in\nchondrocytes and inflammatory responses in synovial fibroblasts through the IL-36R signaling\npathway.\n10. In Figures 6 and 7, the comparison of OA recovery through microneedles (MN) should\ninclude both control groups such as sham-treated mice and intra-articular (i.a.) injection\ntreatment groups for a more comprehensive comparison.\nResponse: Thank you very much for your valuable suggestion. We understand that directly\ncomparing the intra-articular (i.a.) injection treatment groups with the microneedles (MN)\ntreatment groups could make the study more comprehensive. Regarding sham-treated mice and\nintra-articular (i.a.) injection treatment groups, we have conducted relevant experiments in\nSupplementary Figs. 9 and 10, which clearly validate the therapeutic effects of mIL-36Ra in\nexperimental OA. Therefore, the current data sufficiently demonstrate the potential of mIL-\n36Ra in OA treatment.\nIn this study, we emphasize MN as a non-invasive drug delivery method, highlighting its\npotential application in OA treatment. We particularly focus on the clinical advantages it may\noffer, such as higher patient compliance, reduced pain, and easier administration. Compared to\nintra-articular injections, this transdermal delivery approach could be more advantageous in\npractical applications. Additionally, it may be difficult to typeset all the images by adding more\ncontrol groups. We greatly value your suggestion and will consider designing more\ncomprehensive control groups in future studies, particularly when comparing the efficacy of\ndifferent delivery methods.\nReviewer #3 (Remarks to the Author):\nThe authors present an interesting manuscript on skin aging and bone health, mediated by skin\nderived, circulating levels of IL-36 receptor agonists that seem to have a systemic effect. This\nis a fascinating new aspect of skin aging having a systemic effect.\nMajor\nAs far as aspects related to proteomics and transcriptomics are concerned, did the authors use\nadjusted p values for multiple testing? Which correction for multiple testing correction did they\nuse for proteomics and transcriptomics?\nResponse: We sincerely appreciate your valuable suggestions regarding the proteomic and\ntranscriptomic data analyses. We have added some detailed explanations of the significance\nanalysis strategies we applied, based on the data characteristics and sample replicates of\ndifferent experiments in revised Methods and Materials section.\n1. Proteomic analysis in Fig. 2a:\nFor proteomic analysis, we used a combination of unadjusted p-values (< 0.05, determined by\nindependent-sample t-tests) and fold change (FC) thresholds (> 1.5 for upregulation and < 1/1.5\nfor downregulation) to identify significantly differentially expressed proteins, balancing\nstatistical and biological relevance. Multiple testing correction (e.g., FDR or Bonferroni\ncorrection) was not performed. Unadjusted p-values were employed as supportive evidence\nalongside fold change values to ensure biological significance.\n2. Transcriptomics in Fig. 3g-i and Supplementary Fig. 8a:\nFor transcriptomics, we used DESeq2 software to perform differential expression analysis and\ninitially identified differentially expressed genes based on unadjusted p-values. For subsequent\nenrichment analyses (e.g., GO and KEGG), we applied the Benjamini-Hochberg method to\ncorrect the p-values for false discovery rate (FDR) using built-in functions, resulting in q-values.\nA q-value ≤ 0.05 was used as the significance threshold to ensure both biological and statistical\nrelevance of the results.\nMinor\nIn the introduction, the authors might want to explain a bit more why they think their specific\nmodel is an appropriate model reflecting human skin aging.\nResponse: We thank the reviewer for the valuable comments. In this study, we used two mouse\nskin aging models to reflect the characteristics of human skin aging. First, we employed the D-\nGal topical mouse model to simulate the process of human skin aging. D-Gal has been shown\nto induce oxidative stress, accelerate cellular senescence, and trigger inflammatory responses,\nleading to pathological changes in mouse skin similar to human aging. Specifically, D-Gal\npromotes collagen glycation, reduces skin elasticity and moisture content, and increases the\nexpression of senescence markers such as p16. Furthermore, the D-Gal model is widely used\nin skin aging research and effectively reflects the biological processes of human aging through\ninduced oxidative stress and collagen damage.\nSecond, regarding the mouse model of conditional Zmpste24 knockout in skin\nkeratinocytes, we applied AAV-Cre virus to the skin of Zmpste24 fl/fl mice, which resulted in\nabnormal accumulation of prelamin A, a pathology similar to that observed in human skin aging.\nZmpste24 knockout mice are an accelerated aging model, resembling human Hutchinson-\nGilford Progeria Syndrome, and Zmpste24 deletion in articular chondrocytes promotes their\nsenescence. These findings highlight the critical role of Zmpste24 in cellular aging. Therefore,\nknocking out Zmpste24 in skin keratinocytes allows us to model key biological features of skin\naging and explore its potential impact on systemic inflammation and OA progression.\nWe have incorporated the revised content in the final paragraph of the introduction:\n(lines 65-67) In this study, we constructed skin aging models in mice using two approaches:\nchemical (D-Gal) and genetic (Zmpste24 knockout), and these two specific models can\nreasonably reflect human skin aging5, 6. We confirmed the link between aged skin and OA\nprogression while demonstrated that aged skin keratinocytes are the main source of IL-36\nagonists inflammatory mediators that promote OA progression.\nReferences\n1. Fan A, et al. Inhibition of fibroblast activation protein ameliorates\ncartilage matrix degradation and osteoarthritis progression. Bone Res 11, 3\n(2023).\n2. Choi WS, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism\nregulates osteoarthritis. Nature 566, 254-258 (2019).\n3. Lorenz J, Grassel S. Experimental osteoarthritis models in mice. Methods Mol\nBiol 1194, 401-419 (2014).\n4. Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial\nmeniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis\nCartilage 15, 1061-1069 (2007).\n5. Varela I, et al. Accelerated ageing in mice deficient in Zmpste24 protease\nis linked to p53 signalling activation. Nature 437, 564-568 (2005).\n6. Umbayev B, Askarova S, Almabayeva A, Saliev T, Masoud AR, Bulanin D.\nGalactose-Induced Skin Aging: The Role of Oxidative Stress. Oxid Med Cell\nLongev 2020, 7145656 (2020)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Dear authors,\nThank you very much for submitting this new version of the manuscript and for the significant effort you have made to improve it and respond to all the questions and comments raised by this reviewer. You have addressed many of the questions appropriately. However, my main criticism of the manuscript remains unresolved.\nMy primary concern is that the connection between what happens in the skin and what happens in the joint is not convincingly demonstrated, nor is it clearly shown that what occurs in the skin is the cause of knee OA.\nThe authors suggest that \"their results reveal that IL-36 agonists are age-related systemic inflammatory factors released from skin to joints and contribute to OA development.\" This statement is not consistent with the levels of IL-36 agonists in serum and synovial fluid of the patients. There is a higher concentration of IL-36 agonists in synovial fluid than in serum, which is generally explained by local synthesis of IL-36 agonists in the joint tissues. To support the authors' hypothesis, one would expect a higher concentration of IL-36 in the skin and serum than in the synovial fluid. Based on the data presented, what can be concluded is that IL-36 agonists participate in the OA process—something that has already been previously demonstrated.\nAuthors also generated microneedles loaded with mouse recombinant IL-36Ra protein or spesolimab, inserted them directly into skin to sustainably inhibit IL-36R signaling in epidermal keratinocytes, which both, with some dudes, attenuated OA progression in male mice. How do the authors know that this is an effect exclusively occurring in the skin? This effect could be because the mouse recombinant IL-36Ra protein or spesolimab reaches the joint and acts on chondrocytes and synoviocytes.\nRegarding the histologic differences between groups, these are often very weak, mainly OARSI score, subchondral bone, osteophytes and even synovial. Authors recognize that the evaluation of subchondral bone sclerosis and osteophyte formation often does not show statistically significant differences, and they suggest that a possible explanation is the animal model used in this manuscript.\nOverall, histological results (pictures) of the OA animal model are unclear and unconvincing. Differences between groups are often very weak, mainly OARSI score, subchondral bone, osteophytes and even synovial. To clearly support their hypothesis, the authors must convincingly demonstrate that the effects of IL-36 agonists in the joint are due to those derived from the skin and not from IL-36 synthesized locally in the joint.\nReferences 24 and 42 are duplicated.", "reviewer_2": "The authors have thoroughly addressed the comments from the first round of review, including the major revision previously requested. The revised manuscript reflects the feedback from all reviewers in a comprehensive and thoughtful manner. The quality and content of the revisions are appropriate for the standards of this journal. I recommend acceptance of the manuscript.", "reviewer_3": "The authors have responded to all the reviewers' comments."}, "author_response": "Response\nReviewer #1 (Remarks to the Author):\nDear authors,\nThank you very much for submitting this new version of the manuscript and for the significant\neffort you have made to improve it and respond to all the questions and comments raised by\nthis reviewer. You have addressed many of the questions appropriately. However, my main\ncriticism of the manuscript remains unresolved.\nMy primary concern is that the connection between what happens in the skin and what happens\nin the joint is not convincingly demonstrated, nor is it clearly shown that what occurs in the\nskin is the cause of knee OA.\nThe authors suggest that \"their results reveal that IL-36 agonists are age-related systemic\ninflammatory factors released from skin to joints and contribute to OA development.\" This\nstatement is not consistent with the levels of IL-36 agonists in serum and synovial fluid of the\npatients. There is a higher concentration of IL-36 agonists in synovial fluid than in serum, which\nis generally explained by local synthesis of IL-36 agonists in the joint tissues. To support the\nauthors' hypothesis, one would expect a higher concentration of IL-36 in the skin and serum\nthan in the synovial fluid. Based on the data presented, what can be concluded is that IL-36\nagonists participate in the OA process —something that has already been previously\ndemonstrated.\nResponse: We sincerely appreciate your recognition of our efforts in revising the manuscript\nand addressing the reviewers’ comments. We take your critiques very seriously and have\ncarefully re-evaluated the issues you raised. In response to your key concern regarding whether\n“IL-36 agonists are age-related systemic inflammatory factors released from skin to joints and\ncontribute to OA development,” we have conducted additional analyses and experiments, and\nwe address your concerns as follows:\nFirst, we fully understand your scientific concern that “the concentration of IL-36 agonists\nin synovial fluid is higher than in serum,” which seems inconsistent with the hypothesis that\nIL-36 agonists are released from the skin into circulation and subsequently reach the joint. To\nclarify this, we re-analyzed our original data by expanding the sample size and excluding OA\npatients under the age of 70. The new data revealed that in this elderly group, the majority of\npatients (13/15) exhibited higher concentrations of IL-36 agonists in serum than in synovial\nfluid. This supports the hypothesis that IL-36 agonists may act as age-related systemic\ninflammatory mediators, potentially released from the skin into the circulation and\nsubsequently affecting the joint. These updated data have been included in the revised\nmanuscripts and are highlighted in yellow:\nWe also found positive correlations of elevated expression of IL-36 agonists between serum\nand synovial fluid in aged OA patients, with IL-36α concentrations 359.27 ± 21.78 pg/mL in\nserum and 337.01 ± 15.36 pg/mL in synovial fluid, IL-36β concentrations 376.32 ± 18.83\npg/mL in serum and 361.26 ± 18.97 pg/mL in synovial fluid, IL-36γ concentrations 546.15 ±\n35.58 pg/mL in serum and 535.12 ± 10.25 pg/mL in synovial fluid. The determination\ncoefficients (R²) of IL-36α, β, and γ were 0.4716, 0.4660, and 0.3210, respectively (Fig. 2o).\nAuthors also generated microneedles loaded with mouse recombinant IL-36Ra protein or\nspesolimab, inserted them directly into skin to sustainably inhibit IL-36R signaling in epidermal\nkeratinocytes, which both, with some dudes, attenuated OA progression in male mice. How do\nthe authors know that this is an effect exclusively occurring in the skin? This effect could be\nbecause the mouse recombinant IL-36Ra protein or spesolimab reaches the joint and acts on\nchondrocytes and synoviocytes.\nResponse: We do not rule out the possibility that IL-36 family members may also be locally\nexpressed in joint tissues. However, previous studies and our own data suggest that\nkeratinocytes in the skin are one of the main sources of IL-36 agonists (Supplementary Figure\n5). To further validate that the site of IL-36R inhibition was indeed the skin, we assessed the\ntissue-specific responses following microneedle delivery of spesolimab. Our updated results\nshowed that IL-36 agonist levels were markedly reduced in both the skin and serum after\nspesolimab microneedle treatment, while the serum concentration of spesolimab showed a\nslight increase without reaching statistical significance (Supplementary Figure 12g–i). In\naddition, spesolimab treatment showed no significant toxicity to major organs, as indicated by\nH&E staining and organ coefficient (Supplementary Figure 12e–f).\nSimilar results were observed with microneedle delivery of recombinant IL-36Ra protein\n(Figure 6f–h, Supplementary Figure 11c–d).\nThese data collectively suggest that the microneedles primarily exert their IL-36R blocking\neffect locally in the skin. That said, we acknowledge that a certain degree of systemic absorption\nand potential action within the joint cannot be completely excluded, and this limitation has been\nfurther elaborated in the revised Discussion.\nRegarding the histologic differences between groups, these are often very weak, mainly OARSI\nscore, subchondral bone, osteophytes and even synovial. Authors recognize that the evaluation\nof subchondral bone sclerosis and osteophyte formation often does not show statistically\nsignificant differences, and they suggest that a possible explanation is the animal model used\nin this manuscript.\nOverall, histological results (pictures) of the OA animal model are unclear and unconvincing.\nDifferences between groups are often very weak, mainly OARSI score, subchondral bone,\nosteophytes and even synovial. To clearly support their hypothesis, the authors must\nconvincingly demonstrate that the effects of IL-36 agonists in the joint are due to those derived\nfrom the skin and not from IL-36 synthesized locally in the joint.\nReferences 24 and 42 are duplicated.\nResponse: Regarding the relatively mild histological differences between groups, our study\nfocused on chondrocytes and synovial fibroblasts, which are the main target cells of IL-36\nagonists. Accordingly, significant differences were observed in OARSI scores and synovitis\ngrading. However, in a few groups, the changes in subchondral bone sclerosis and osteophyte\nformation were not statistically significant. We hypothesize that this may be not only due to the\nDMM model used but also related to the need for longer treatment durations to observe\nimprovements in subchondral bone changes. These considerations have now been included in\nthe revised Discussion section. Moreover, we have re-evaluated and updated the scoring of\nsubchondral bone sclerosis and osteophyte formation in the revised manuscript.\nLastly, we appreciate your attention to detail regarding reference formatting. As you pointed\nout, references 24 and 42 were duplicated. We have carefully reviewed the reference list,\nremoved the duplicated entry, and updated the numbering accordingly.\nThank you again for your valuable comments and constructive feedback, which have\nprompted us to more thoroughly examine and improve our work. We sincerely hope that our\nrevisions address your concerns.\nReviewer #2 (Remarks to the Author):\nThe authors have thoroughly addressed the comments from the first round of review, including\nthe major revision previously requested. The revised manuscript reflects the feedback from all\nreviewers in a comprehensive and thoughtful manner. The quality and content of the revisions\nare appropriate for the standards of this journal. I recommend acceptance of the manuscript.\nResponse: We sincerely thank you for your thorough review and your recommendation for\nacceptance, and truly appreciate your recognition of the efforts we made to address the\ncomments from the first round. Your positive feedback gives us great motivation and\nconfidence to continue improving the quality of our work. Thank you again for your time and\nsupport.\nReviewer #3 (Remarks to the Author):\nThe authors have responded to all the reviewers' comments.\nResponse: We sincerely thank you for your recognition. Your affirmation is truly encouraging for\nus, and we are grateful for the time and effort you dedicated to reviewing our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Thank you very much for sending this new version of the manuscript. Now, the new experiments and re-analysis of the results improve the scientific data of the paper. Comments and limitations at the interpretation of the results added in the discussion have also big value.", "reviewer_4": "The statistical methods used in the manuscript are appropriate. One minor comment is that for proteomics , it’s more standard to phrase as “differentially abundant” instead of “differentially expressed”. Otherwise, the RNA-seq and LC-MS/MS proteomics analyses are clearly described, and the use of the Benjamini–Hochberg procedure to control the false discovery rate is appropriate.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response\nReviewer #1 (Remarks to the Author):\nThank you very much for sending this new version of the manuscript. Now, the new\nexperiments and re-analysis of the results improve the scientific data of the paper. Comments\nand limitations at the interpretation of the results added in the discussion have also big value.\nResponse: We sincerely thank you for your thorough reviews across the previous rounds. We\nare grateful that you find the new experiments and re-analyses have improved the scientific\nstrength of the paper, your guidance has been instrumental in refining this work.\nReviewer #4 (Remarks to the Author):\nThe statistical methods used in the manuscript are appropriate. One minor comment is that for\nproteomics, it’s more standard to phrase as “differentially abundant” instead of “differentially\nexpressed”. Otherwise, the are clearly described, and the use of the Benjamini–Hochberg\nprocedure to control the false discovery rate is appropriate.\nResponse: We sincerely appreciate your recognition of our statistical methods and the\ndescriptions of the RNA-seq and LC–MS/MS proteomics analyses. We are also grateful for\nyour constructive suggestion to use “differentially abundant” terminology; accordingly, we\nhave revised the manuscript to adopt “differentially abundant proteins” throughout in place\nof “differentially expressed protein.”\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank you for co-reviewing this manuscript as part of the Nature Communications\nEarly Career Researcher peer-review initiative. We are grateful for your time, constructive feedback,\nand contribution to the review process."}]

Decision letter