PNAS PLUS

JAK inhibition alleviates the cellular senescenceassociated secretory phenotype and frailty in old age Ming Xua, Tamara Tchkoniaa, Husheng Dinga, Mikolaj Ogrodnika,b, Ellen R. Lubbersa, Tamar Pirtskhalavaa, Thomas A. Whitea, Kurt O. Johnsona, Michael B. Stouta, Vojtech Mezeraa, Nino Giorgadzea, Michael D. Jensena, Nathan K. LeBrasseura, and James L. Kirklanda,1 a Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN 55905; and bNewcastle University Institute for Ageing, Newcastle University, Newcastle Upon Tyne, NE4 5PL, United Kingdom

Chronic, low grade, sterile inflammation frequently accompanies aging and age-related diseases. Cellular senescence is associated with the production of proinflammatory chemokines, cytokines, and extracellular matrix (ECM) remodeling proteases, which comprise the senescence-associated secretory phenotype (SASP). We found a higher burden of senescent cells in adipose tissue with aging. Senescent human primary preadipocytes as well as human umbilical vein endothelial cells (HUVECs) developed a SASP that could be suppressed by targeting the JAK pathway using RNAi or JAK inhibitors. Conditioned medium (CM) from senescent human preadipocytes induced macrophage migration in vitro and inflammation in healthy adipose tissue and preadipocytes. When the senescent cells from which CM was derived had been treated with JAK inhibitors, the resulting CM was much less proinflammatory. The administration of JAK inhibitor to aged mice for 10 wk alleviated both adipose tissue and systemic inflammation and enhanced physical function. Our findings are consistent with a possible contribution of senescent cells and the SASP to age-related inflammation and frailty. We speculate that SASP inhibition by JAK inhibitors may contribute to alleviating frailty. Targeting the JAK pathway holds promise for treating age-related dysfunction. JAK/STAT pathway

| cellular senescence | ruxolitinib | interleukin-6 | frailty

hallmark of aging is chronic, low-grade, “sterile” inflammation (1–3). Elevated proinflammatory cytokines and chemokines are closely associated with mortality (4, 5) and with a variety of age-related diseases, including atherosclerosis (6), depression (7), cancers (8), diabetes (9), and neurodegenerative diseases (10, 11). Inflammation also is associated with frailty, a geriatric syndrome characterized by decreased strength and incapacity to respond to stress (2). The underlying mechanisms of age-related chronic inflammation, tissue dysfunction, and frailty remain elusive. Cellular senescence, stable arrest of cell growth in replication-competent cells, is a plausible contributor. Senescence can be induced by a number of stimuli and stresses, including telomere dysfunction, genomic instability, oncogenic and metabolic insults, and epigenetic changes (12). Senescent cells accumulate with aging in the skin (13, 14), liver (15, 16), kidney (17), the cardiovascular system (18), and other tissues in various species (16). The senescence-associated secretory phenotype (SASP), largely comprised of proinflammatory cytokines and chemokines (19, 20), links senescent cells to age-related inflammation and diseases. We found that elimination of senescent cells delayed the onset of age-related phenotypes and enhanced healthspan (21, 22). Therefore, senescent cells and the SASP could play a role in age-related pathologies, particularly those that involve systemic inflammation. The JAK/STAT pathway plays an important role in regulating cytokine production (23, 24). We hypothesized that it may directly affect the SASP. The JAK family has four members: JAK1,

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JAK2, JAK3, and tyrosine kinase 2 (TYK2). JAK1 and 2 are involved in inflammatory signaling and in the action of growth hormone and other endocrine and paracrine signals (25). JAK3 is expressed primarily in blood cells and mediates erythropoietin signaling and immune cell generation (26–29). TYK2 plays an important role in white blood cell function and host defenses (30, 31). A number of inhibitors that target different JAKs have been approved or are in phase I–III studies for treating myelofibrosis (23, 32– 34), acute myeloid leukemia (23), lymphoma (23), and rheumatoid arthritis (23, 35, 36). JAK inhibitors recently have been found to reprogram the SASP in senescent tumor cells, contributing to improved antitumor response (37). Therefore, we investigated the role of the JAK pathway in age-related inflammation and dysfunction. We demonstrate here that senescent preadipocytes, fat cell progenitors, accumulate in adipose tissue with aging and can contribute to adipose tissue inflammation. We found that JAK inhibitors decrease the SASP in preadipocytes and human umbilical vein endothelial cells (HUVECs). They also decrease age-related adipose tissue and systemic inflammation as well as frailty. Our findings provide insights into the possible contribution of senescent cells to age-related inflammation and, in turn, to age-related pathologies, as well as potential therapeutic targets to alleviate age-related dysfunction. Significance A hallmark of aging is chronic sterile inflammation, which is closely associated with frailty and age-related diseases. We found that senescent fat progenitor cells accumulate in adipose tissue with aging and acquire a senescence-associated secretory phenotype (SASP), with increased production of proinflammatory cytokines compared with nonsenescent cells. These cells provoked inflammation in adipose tissue and induced macrophage migration. The JAK pathway is activated in adipose tissue with aging, and the SASP can be suppressed by inhibiting the JAK pathway in senescent cells. JAK1/2 inhibitors reduced inflammation and alleviated frailty in aged mice. One possible mechanism contributing to reduced frailty is SASP inhibition. Our study points to the JAK pathway as a potential target for countering age-related dysfunction. Author contributions: M.X., T.T., and J.L.K. designed research; M.X., T.T., H.D., M.O., E.R.L., T.P., T.A.W., K.O.J., M.B.S., V.M., N.G., and M.D.J. performed research; M.X., T.T., M.O., and N.K.L. analyzed data; and M.X., T.T., and J.L.K. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Y.B.-N. is a guest editor invited by the Editorial Board. Freely available online through the PNAS open access option. 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1515386112/-/DCSupplemental.

PNAS | Published online November 2, 2015 | E6301–E6310

CELL BIOLOGY

Edited by Yinon Ben-Neriah, Hebrew University, Jerusalem, Israel, and accepted by the Editorial Board September 16, 2015 (received for review August 3, 2015)

Results Senescent Cells Accumulate in Adipose Tissue with Aging. Preadipocytes,

also termed “adipose-derived stem cells” or “fat cell progenitors” (38), constitute 15–50% of the cells in adipose tissue and arguably are the most abundant progenitor cell type in humans (39). We first tested whether the number of senescent preadipocytes increases in adipose tissue with aging. We isolated primary preadipocytes from the stromal-vascular fraction of abdominal s.c. fat tissue from healthy old and young human subjects. We stained these cells with histone γH2AX and the cell proliferation marker Ki-67. Cells with γH2AX+ foci and Ki-67− nuclei were considered to be senescent cells. We found that more than 10% of preadipocytes in adipose tissue from old human subjects but less than 4% of preadipocytes in adipose tissue from young subjects were senescent (Fig. 1). Similar results were found when these cells were assayed for cellular senescence-associated β-galactosidase (SABG) (Fig. S1A). Consistent with our data in humans, we found there are more SABG+ cells in adipose tissue from 30-mo-old rats than in adipose tissue from 3-mo-old rats (Fig. S2A). The frequency of γH2AX+ (a marker of DNA damage) cells also was higher in adipose tissue from 30-mo-old rats (Fig. S2C). Levels of p21Cip1 protein, a marker of cellular senescence, were higher in these adipose depots in the old animals (Fig. S2B). Both activated (phosphorylated) JAK1 (Tyr1022/1023) and JAK2 (Tyr1007/1008) were increased in adipose tissue from old rats as well (Fig. S2B). We observed more senescent preadipocytes isolated from the adipose tissue of 30-mo-old rats than from the adipose tissue of 3-mo-old rats (Fig. S1 B and C). In addition, we recently demonstrated that aging is associated with an increased abundance of senescent cells in the adipose tissue of mice (40). Therefore, senescent cells accumulate in fat tissue with aging across several mammalian species. Senescent Preadipocytes Develop a SASP. To test if human primary preadipocytes exhibit a SASP upon becoming senescent, we irradiated preadipocytes from healthy human kidney transplant donors with 10 Gy to induce senescence. After 20 d, more than 70% of preadipocytes became SABG+ (Fig. 2A), with increased p16Ink4a and p21Cip1 mRNA abundance (Fig. 2B). We also induced cellular senescence by serial passage. By passage 27–30, most preadipocytes had stopped proliferating, and more than 80% were SABG+ (Fig. 2A) with increased p16Ink4a and p21Cip1 transcript abundance (Fig. 2B). To test cytokine secretion by these cells, we collected conditioned medium (CM) from senescent and control cells for 24 h. In an addressable laser bead-based multiplex assay, most of the cytokines and chemokines measured were significantly higher in CM from irradiation-induced senescent than from

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control preadipocytes (Table S1). The most highly induced cytokines and chemokines were IL-6 (19-fold), GM-CSF (28fold), granulocyte-colony stimulating factor (G-CSF) (10-fold), chemokine (C-X-C motif) ligand 1 (CXCL-1) (27-fold), macrophage inflammatory protein-1α (MIP-1α) (30-fold), IL-8 (46-fold), monocyte chemotactic protein-1 (MCP-1) (19-fold), regulated on activation, normal T-cell–expressed and –secreted (RANTES) (15fold), and MCP-3 (17-fold) (see Fig. 4B; full results are in Table S1). Similar results were found for IL-6 and MCP-1 proteins in CM by ELISA (Fig. S3A). In serial passage-induced senescent cells, we also detected the induction of secreted cytokines including IL-6, IL-8, MCP-1, and plasminogen activator inhibitor-1 (PAI-1) using the multiplex assay (Fig. S3B). Consistent with the increased secreted cytokines, we found increased mRNA levels for several key SASP components, including IL-1α, IL-1β, IL-6, IL-8, MCP-1, matrix metalloproteinase (MMP)-3, MMP-12, PAI-1, and TNF-α in both irradiation- and serial passage-induced senescent preadipocytes compared with nonsenescent controls (Fig. 2C). Senescent Preadipocytes Induce Inflammation in Adjacent Cells. Next, we tested whether senescent preadipocytes confer inflammation to adjacent healthy cells. If so, this effect would suggest that the SASP can amplify adipose tissue inflammation. Exposure of nonsenescent preadipocytes or adipose tissue from healthy donors to CM from irradiation-induced senescent preadipocytes for 24 h induced the expression of inflammatory markers (Fig. 3 A and C, respectively) in a dose-dependent manner (Fig. S4), compared with exposure to CM from nonsenescent preadipocytes. Infiltration of macrophages into adipose tissue is associated with adipose tissue dysfunction and inflammation (41). To determine whether senescent cells could drive this event, we conducted cell migration assays using THP1 monocytes. Relative to CM from nonsenescent preadipocytes, CM derived from senescent cells caused a more than fourfold increase in THP1 monocyte migration within 6 h (Fig. 3B). Inhibition of JAK Suppresses the SASP in Senescent Preadipocytes.

Given the role of the JAK/STAT pathway in inflammation, we examined whether inhibiting the JAK/STAT pathway blunts the SASP. We treated irradiation-induced senescent preadipocytes for 72 h with various concentrations (0–1 μM) of JAK inhibitor 1, which inhibits all four JAKs. We found that at a concentration of 0.6 μM JAK inhibitor 1 suppressed the mRNA levels of key SASP components, including IL-6, IL-8, and MCP-1, by up to 60%. Increasing the concentration of JAK inhibitor 1 to 1 μM did not further suppress the SASP (Fig. 4A). JAK inhibitor 1 had little effect on the same components in nonsenescent control cells (Fig. S5). In a multiplex assay for cytokines and chemokines,

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Fig. 1. Senescent preadipocytes accumulate in adipose tissue with aging. Primary preadipocytes were isolated from femoral s.c. fat obtained by needle biopsy from four young (31 ± 5 y) and four old (71 ± 2 y) healthy male volunteers and then were stained with γH2AX and Ki-67. (A) Representative images of human cells. (B) The percentages of γH2AX+ and Ki-67− preadipocytes were counted. Results are expressed as mean ± SEM; *P < 0.05; n = 4.

E6302 | www.pnas.org/cgi/doi/10.1073/pnas.1515386112

Xu et al.

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Fig. 2. Senescent preadipocytes develop a SASP. (A) Preadipocytes were isolated from healthy human subjects. Control (CON, Left), irradiated (IRA, Center), and serially passaged (SP, Right) preadipocytes were assayed for cellular SABG activity and stained with DAPI. (B) The relative mRNA abundance of p16 and p21 in control (CON) and irradiation-induced (IRA) and serial passage-induced (SP) senescent preadipocytes is shown. Results are expressed as mean ± SEM; *P < 0.05; n = 7. (C) The relative mRNA abundance of key SASP components in control (CON), irradiation-induced (IRA), and serial passage-induced (SP) senescent preadipocytes is shown. Results are expressed as mean ± SEM; *P < 0.05 compared with nonsenescent controls; n = 5.

we observed that 16 of 32 proteins were significantly lower in the CM from senescent preadipocytes treated with JAK inhibitor 1 (0.6 μM for 72 h; CM collected for the last 24 h) than in the CM from vehicle-treated senescent preadipocytes (Table S1). IL-6 (58%), GM-CSF (68%), G-CSF (60%), IFN-γ–induced protein 10 (IP-10) (92%), CXCL-1 (48%), MIP-1α (90%), IL-8 (62%), MCP-1 (59%), RANTES (52%), and MCP-3 (71%) were among the SASP components most prominently suppressed by JAK inhibitor 1 (Fig. 4B; full results are in Table S1). MMP3 and MMP12 mRNA levels were decreased by 80% when senescent human preadipocytes were treated with JAK inhibitor 1 (Fig. 4C). We also assessed the extent to which JAK inhibitor 1 affected the release of key SASP components in serial passage-induced senescent preadipocytes. Similar to our observations in irradiation-induced senescent cells, CM from passage-induced senescent cells treated with JAK inhibitor 1 contained less IL-6, IL-8, and MCP-1 than CM from vehicle-treated cells (Fig. S3B). Expression levels of several key SASP components also were reduced in passage-induced senescent cells treated with JAK inhibitor 1 (Fig. 4D). Endothelial cells are a major population of cells in adipose tissue and elsewhere, and we found that senescent endothelial cells accumulate in adipose tissue (42). JAK inhibitor 1 suppressed the SASP in irradiation-induced HUVECs (Fig. S6), suggesting that inhibition of the JAK/STAT pathway could suppress the SASP in multiple senescent cell types. Xu et al.

To exclude the possibility of nonspecific effects of JAK inhibitor 1, we treated irradiation-induced senescent preadipocytes with two other JAK inhibitors, momelotinib (CYT387) and INCB18424, both of which specifically inhibit JAK1 and JAK2. These two specific inhibitors blunted the SASP similarly to JAK inhibitor 1 (Fig. 4E). STAT3 is a STAT family member that induces and maintains an inflammatory microenvironment by regulating a range of inflammatory cytokines and mediators, including the SASP components IL-6, IL-8, PAI-1, MCP-1, and GM-CSF (24, 43). We observed that phosphorylated STAT3 (Tyr705) was elevated in senescent preadipocytes and was substantially decreased upon treatment with JAK inhibitor 1 (Fig. 4F), consistent with a role for JAK1/2 activation in the SASP. All three JAK inhibitors target both JAK1 and JAK2. To distinguish the role of JAK1 from that of JAK2, we used siRNA to knock down either JAK1 or JAK2 expression in senescent preadipocytes. We used two different siRNAs against the same gene transcripts to exclude off-target effects of these siRNAs. We were able to knock down JAK1 and JAK2 mRNA levels by about 85% and 80%, respectively. Reduced expression of either JAK1 or JAK2 led to SASP inhibition (Fig. 5). Together, these findings indicate that both JAK1 and JAK2 have roles in regulating the SASP. PNAS | Published online November 2, 2015 | E6303

CELL BIOLOGY

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Fig. 3. Senescent preadipocytes induce preadipocyte inflammation, macrophage migration, and adipose tissue inflammation. (A) Preadipocytes were isolated from healthy human subjects and treated with CM collected from control (CON) or senescent (SEN) preadipocytes for 24 h. Then RNA was collected, and real-time PCR was performed. (B) THP-1 cells were placed in inserts in the top chamber, and CM from control (CON) or senescent (SEN) preadipocytes was introduced into the lower chamber. Cell migration was tested following 6 h of exposure using the CyQUANT cell proliferation assay. Results are shown as the fold-change of cell number in each treatment vs. the control blank CM group. (C) Subcutaneous adipose tissue was isolated from healthy human subjects and treated with CM collected from control (CON) or senescent (SEN) preadipocytes for 24 h. Then RNA was collected, and real-time PCR was performed. The results were obtained from six sets of CM from the preadipocytes of six subjects and are expressed as mean ± SEM. *P < 0.05.

JAK Inhibition Decreases Inflammation Induced by Senescent Cells.

Because JAK inhibitors suppress the SASP, we tested if SASP suppression reduces the inflammation and chemoattraction caused by senescent cell CM. Nonsenescent preadipocytes expressed significantly less IL-6, MCP-1, and PAI-1 when incubated with CM generated from senescent cells treated with JAK inhibitor than when incubated with CM generated from vehicle-treated senescent cells. Interestingly, IL-1α, IL-1β, and IL-8 expression did not change with JAK inhibitor treatment (Fig. 6A), suggesting that the SASP is segmentally suppressed by JAK inhibitors. Macrophage migration was reduced in response to CM from senescent preadipocytes treated with a JAK inhibitor compared with CM from vehicletreated senescent cells (Fig. 6B). Because the JAK inhibitor was present in CM from the senescent cells treated with JAK inhibitor, we tested whether the addition of a JAK inhibitor to CM from senescent preadipocytes not treated with a JAK inhibitor could prevent SASP-induced inflammation. We treated nonsenescent preadipocytes with CM from irradiation-induced senescent preadipocytes for 24 h in the presence of vehicle or 0.6 μM JAK inhibitor 1 (both vehicle and JAK inhibitor 1 had been placed in a 37 °C incubator for 24 h to mimic the CM collection process). JAK inhibition had little effect on reducing target cell inflammation induced by CM from senescent preadipocytes (Fig. 6C). Therefore, JAK inhibitors E6304 | www.pnas.org/cgi/doi/10.1073/pnas.1515386112

alleviated inflammation mainly by suppressing the SASP in senescent cells rather than by acting on nonsenescent cells to prevent the inflammation caused by the action of SASP components. Inhibition of JAK/STAT Pathways Decreases Systemic and Adipose Tissue Inflammation in Old Mice. Aging is associated with chronic

low-grade sterile inflammation (1–3). JAK inhibition partially rescues the chronic low-grade sterile inflammation caused by senescent preadipocytes in adipose tissue in vitro. Therefore, we examined whether inhibition of the JAK pathway reduced systemic inflammation in aged mice. We treated 24-mo-old C57BL/6 male mice with ruxolitinib (INCB18424), a selective JAK1/2 inhibitor approved by the Food and Drug Administration, or vehicle (DMSO) for 10 wk. We measured the levels of an array of cytokines and chemokines in sera from these old mice and from eight young (6-mo-old) mice as controls (Table S2). Seven targets were increased significantly with age: IL-6, G-CSF, IP-10, CXCL1, MIP-1β, IL-17, and chemokine (C-C motif) ligand-11 (CCL-11). JAK inhibitor treatment significantly decreased five of these targets as well as GM-CSF, CXCL9, and IL-15, which were not significantly increased with age (Fig. 7A; full results are in Table S2). Notably, IL-6, GM-CSF, G-CSF, IP-10, and CXCL-1 levels were suppressed by JAK inhibitor both in CM from senescent Xu et al.

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Fig. 4. JAK inhibitor suppresses the SASP in senescent preadipocytes. (A) Irradiation-induced senescent preadipocytes were treated with different concentrations of JAK inhibitor 1 for 72 h. Then RNA was collected, and real-time PCR was performed. Results are expressed as mean ± SEM; *P < 0.05 compared with the vehicle (Veh) group; n = 3. (B) CM was collected from control adipocytes (CON), senescent adipocytes treated with DMSO (SEN), and senescent preadipocytes treated with 0.6 μM JAK inhibitor 1 (SEN+JAKi). Cytokine protein levels in CM were measured by multiplex assay and were normalized by cell numbers. Selected cytokines levels are shown as the percentage of SEN for primary preadipocytes from each human subject. Results are expressed as mean ± SEM; *P < 0.05 compared with SEN; n = 6. (C) Irradiation-induced senescent preadipocytes were treated with 0.6 μM JAK inhibitor 1 for 72 h. Then RNA was collected, and real-time PCR was performed. Results are expressed as mean ± SEM; *P < 0.05; n = 6. (D) Serial passage-induced senescent preadipocytes were treated with 0.6 μM JAK inhibitor 1 for 72 h. Then RNA was collected, and real-time PCR was performed. Results are expressed as mean ± SEM; *P < 0.05; n = 6. (E) Irradiation-induced senescent preadipocytes were treated with different JAK inhibitors, including JAK inhibitor 1 (Jak1), momelotinib (CYT), and ruxolitinib (INCB), each at 1 μM for 72 h. Then RNA was collected, and real-time PCR was performed. Results are expressed as mean ± SEM; *P < 0.05 compared with the vehicle (Veh) group; n = 5. (F) Radiation-induced senescent preadipocytes were treated with 0.6 μM JAK inhibitor 1 for 72 h. Then cell lysates were prepared. Phosphorylated STAT3 (P-STAT3) (Tyr705), total STAT3, and GAPDH protein abundance are shown. n = 4; representative images are depicted.

preadipocytes (Fig. 4B) and in serum from aged mice (Fig. 7A). IL-6, G-CSF, and CXCL-1 levels were induced by both cellular senescence (Fig. 4B) and aging (Fig. 7A). To test whether the decreased levels of cytokines were related to reduced numbers of white blood cells, we performed complete blood cell counts in these mice. We observed that 10 wk of treatment with INCB18424 did not alter number of peripheral blood immune cells in aged mice (Table S3). Adipose tissue is a major source of the cytokines in aged animals (44, 45). We found that the expression of several inflammatory markers and matrix metalloproteinases in adipose tissue, including IL-6, TNF-α, MMP-3, MMP-12, and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1 or F4/80), a macrophage marker, was decreased in the INCB18424treated group, (Fig. 7B). This decrease in Emr-1 is consistent with the reduction in senescent CM-induced macrophage diapedesis we found following JAK inhibition in vitro (Fig. 6B). In addition, we found increased phosphorylated JAK2 (Tyr1007/1008) and STAT3 (Tyr705) in aged adipose tissue along with p21Cip1. Only phosphorylated STAT3 was significantly reduced by JAK inhibitor treatment (Fig. 7 C and D). This finding suggests that STAT3 plays a role in age-related adipose tissue inflammation. JAK Inhibition Enhances Physical Activity in Aged, Frail Mice. The prevalence of frailty increases in old age. Phenotypic characteristics Xu et al.

of frailty include reduced muscle strength, poor endurance, and low physical activity (46). Increased serum IL-6 is associated with decreased physical activity and frailty in older people (47–50). We tested whether the decreased abundance of IL-6 and other inflammatory cytokines in response to JAK1/2 inhibitors is associated with enhanced physical function in aged mice. Before treatment, there were no differences in baseline measures of physical activity, hanging endurance, or grip strength between groups of mice randomized to treatment with either vehicle or the JAK inhibitor. After 10 wk of treatment, administration of the JAK1/2 inhibitor enhanced physical activity (Fig. 8A), including rearing [reduced rearing is a marker of frailty (51)] (Fig. 8B) and ambulation counts (Fig. 8C). The administration of the JAK1/2 inhibitor also improved hanging endurance (Fig. 8D) and grip strength (Fig. 8E) in these aged mice. Moreover, inhibition of the JAK pathway partially restored the agerelated decline in coordination (Fig. 8F). Body weight and food intake remained similar between the vehicle-treated mice and those treated with the JAK inhibitor. Thus, JAK inhibitor treatment enhanced overall physical function in frail, aged mice. Discussion Systemic inflammation increases with advancing age (52). The exact mechanism for this increase is still not completely understood. Potential mechanisms include a deregulated immune PNAS | Published online November 2, 2015 | E6305

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Fig. 5. Knockdown of either JAK1 or JAK2 suppresses the SASP in senescent preadipocytes. (A, Left) Irradiation-induced senescent preadipocytes were transduced with nontargeting siRNA (siCON) or either of two JAK1 siRNAs (siJAK1-a and siJAK1-b). (B, Left) Irradiation-induced senescent preadipocytes were transduced with nontargeting siRNA (siCON) or one of two JAK2 siRNAs (siJAK2-a and siJAK2-b). RNA was collected 48 h after transduction, and real-time PCR was performed to detect mRNA of key SASP components. Results are expressed as mean ± SEM; *P < 0.05 compared with the siCON group; n = 4 for each group. Total JAK1, total JAK2, and GAPDH protein levels are shown also. (A and B, Right) Representative images are shown.

system (53) or altered redox homeostasis (54). Increased numbers of T cells and inflammatory macrophages also are observed in aged adipose tissue (44). Our findings support the possibility that senescent cells contribute to age-related systemic inflammation. Evidence for this notion includes the following points. (i) Senescent cells accumulate in aged adipose and other tissues in the mouse (40), rat, and human. (ii) Senescent preadipocytes develop a proinflammatory SASP that can induce inflammation in adjacent healthy cells and whole adipose tissue. (iii) p16Ink4apositive senescent cells were the main cause of increased IL-6 expression in the stromal vascular fraction of adipose tissue digests from older progeroid mice (21). The stromal vascular fraction is, in turn, the principal site of cytokine release by adipose tissue (44). (iv) Inhibiting the JAK-STAT pathway suppresses the SASP in preadipocytes and endothelial cells as well as SASP-induced adipose tissue inflammation in vitro. We found that JAK inhibition also attenuates age-related adipose tissue and systemic inflammation together with frailty in vivo. Collectively, these findings suggest that targeting senescent cells could be a promising way to alleviate age-related chronic inflammation of adipose tissue. E6306 | www.pnas.org/cgi/doi/10.1073/pnas.1515386112

In this study, we mainly used human primary preadipocytes to study cellular senescence and the SASP because fat tissue is a major source of circulating inflammatory cytokines in aged populations (44, 45) and because fat tissue is the largest organ in most humans and preadipocytes thus are perhaps the most abundant progenitor cell type in humans (39). It is likely that senescent cells in other tissues contribute to age-related inflammation as well. Consistent with this possibility, we found that JAK inhibitors decreased the SASP in both preadipocytes (Fig. 6) and endothelial cells (Fig. S6), cell types from two distinct lineages. This finding is consistent with the observation that JAK inhibitors can reprogram the SASP in senescent tumor cells, leading to improved antitumor immune responses (37). There are three potential approaches for targeting senescent cells. One is to prevent them from arising by disabling p16- or p53-related processes or other upstream mechanisms that drive the generation of cellular senescence. This approach, however, is likely to induce cancer (55). The second is to eliminate senescent cells that already have formed. The third is to blunt the proinflammatory nature of the SASP. Mounting evidence suggests that senescent cells can have both harmful and beneficial effects (56). Xu et al.

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Therefore, partial suppression of the SASP seems to be a reasonable option, particularly when short-term alleviation of agerelated dysfunction might be indicated. Several drugs, including glucocorticoids (57), metformin (58), and rapamycin (59), have been found to inhibit the SASP. Inhibition of the JAK/STAT pathway appears to be a viable option for the following reasons. The JAK/STAT pathway regulates the expression of a number of proinflammatory cytokines (24, 43). We found that phosphorylated JAK1 and -2 levels are increased in aged adipose tissue (Fig. S2B). Furthermore, several JAK inhibitors are being used to treat cancers and hematological and rheumatologic conditions in humans (33, 36). However, few studies have investigated their potential benefits in old age. Here, we found that these inhibitors suppress particular SASP components by 40–60% within 3 d, under conditions in which they had little evident effect on nonsenescent cells (Fig. S5). Importantly, JAK inhibitor treatment for 10 wk decreased systemic inflammation and improved physical function in frail, aged mice. Consistent with this result, a recent human trial using roxolitinib for myelofibrosis suggested that JAK inhibition can increase activity in human subjects and reduce frailty-like constitutional symptoms related to hematological disease (33, 60). Thus, inhibition of the JAK pathway is a potentially promising strategy for alleviating age-related dysfunction. IL-6, a major SASP component, signals through the JAK/ STAT pathway (61) and has been shown to be critical in stabilizing cellular senescence (62). Therefore, we tested whether JAK inhibition reduces cellular senescence. However, we did not observe a significant alteration in senescent cell abundance in either in vitro or in vivo models. This lack of effect could be related to the possibility that we treated cells or aged mice with JAK inhibitor Xu et al.

once cellular senescence was established. Because established senescent cells already are present in older individuals, JAK inhibition could prove to be a way to alleviate frailty or cellular senescence-related chronic diseases in late life, points that warrant further preclinical and clinical study. Roxolitinib can have side effects, including anemia and thrombocytopenia (33, 63), in humans who have myelofibrosis. We did not observe an alteration of red blood cell numbers or hemoglobin levels in old mice treated with roxolitinib (Table S3), although platelet counts tended to be reduced (not significantly; n = 9). Roxolitinib treatment had minimal effect on the numbers of peripheral blood immune cells in aged mice (Table S3). One reason is that roxolitinib is a selective JAK1/2 inhibitor that has much less effect on JAK3 and TYK2 (64), the two JAK family members associated with immune cell generation and function (28–31). Notably, in humans treated with roxolitinib for 24 wk for myelofibrosis, major infections were no more frequent than in the placebo group: only one case of clostridial infection was found in the ruxolitinib group, and one case of staphylococcal infection was found in the placebo group (33). More work is needed to uncover potential side effects of JAK1/2 inhibition in older populations. Possibly, the alleviation of frailty by JAK1/2 inhibition that we observed occurs through effects on cells other than senescent cells, such as immune cells or the brain. However, we did not find altered peripheral white blood cell counts (Table S3), and at least one test of brain function remained unaffected in aged mice treated with roxolitinib (Fig. S7). In addition, roxolitinib treatment did not alter the physical activity of 6-mo-old mice (Fig. S8). Other mechanisms for the effects of JAK1/2 inhibition on frailty merit future investigation. However, it appears reasonable to PNAS | Published online November 2, 2015 | E6307

CELL BIOLOGY

Fig. 6. JAK inhibition decreases inflammation induced by senescent cells. (A) Preadipocytes were isolated from healthy human subjects and were treated for 24 h with CM collected from control (CON) preadipocytes, senescent (SEN) preadipocytes, or senescent preadipocytes treated with JAK inhibitor 1 (SEN+JAKi). Then RNA was collected, and real-time PCR was performed. (B) THP-1 cells were placed in inserts in the top chamber, and CM from different groups was introduced into the lower chamber. Cell migration was tested following 6 h of exposure using the CyQUANT cell-proliferation assay. Results are shown as the fold-change of cell number in each treatment vs. the control CM group. (C) Preadipocytes isolated from healthy human subjects were treated for 24 h with CM collected from senescent preadipocytes in the presence of DMSO (SEN-CM) or 0.6 μM JAK inhibitor (SEN-CM+JAKi). Then RNA was collected, and real-time PCR was performed. Results are expressed as mean ± SEM; n = 4.

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Fig. 7. JAK inhibition decreases systemic and adipose tissue inflammation in old mice. Twenty-four–month–old male mice were treated with vehicle (Old Veh) or ruxolitinib (Old INCB) for 10 wk. Six-month-old male mice (Young) were used as young control group. (A) Serum levels of cytokines were measured by multiplex assay. Selected cytokines levels are shown as the fold change over the average level of Young group. Results are expressed as mean ± SEM; *P < 0.05 compared with Young; #P < 0.05 compared with Old Veh; n = 8. (B) RNA of inguinal adipose tissue from old mice was collected, and real-time PCR was performed. Results are expressed as mean ± SEM; *P < 0.05; n = 8. (C) Cell lysates were prepared from inguinal adipose tissue. The abundance of phosphorylated JAK2 (P-JAK2) (Tyr1007/1008), total JAK2, P-STAT3 (Tyr705), total STAT3, p21, and GAPDH protein abundance is shown. (D) These signals were quantified by densitometry using ImageJ. The ratios of phosphorylated to total STAT3 (STAT3 P/T), phosphorylated to total JAK2 (Jak2 P/T), and total p21 level to GAPDH (P21/GAPDH) are expressed as mean ± SEM; *P < 0.05; n = 4.

hypothesize that cellular senescence is one potential mechanism associated with aging-related adipose tissue inflammation. Fat tissue inflammation contributes to elevated circulating cytokines in old age. These increased cytokines, in turn, are associated with or can cause frailty in older humans and experimental animals. Furthermore, genetic or pharmacological clearance of senescent

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cells in older mice alleviated frailty (21, 22), much as JAK inhibitors did in this study and in humans with myeloproliferative disorders (63). In summary, our study suggests that the JAK1/2 pathway plays an important role in the SASP and could be a target for future interventions to alleviate age-related dysfunction.

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Fig. 8. JAK inhibition enhances physical function in aged, frail mice. Twenty-four–month–old male mice were monitored using CLAMS before and after 10 wk of vehicle (CON) or ruxolitinib (INCB) treatment. (A–C) Total activity (A), rearing activity (B), and ambulation (C) were analyzed. Results are shown for both light (Day) and dark (Night) cycles. (D and E) Hanging test (D) and grip strength (E) tests also were performed on these animals. The RotaRod test was performed 8 wk after treatment in parallel with nine 6-mo-old male mice (Young). Results are expressed as mean ± SEM; *P < 0.05; n = 8 for A–C; n = 9 for D–F.

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Xu et al.

Cell Culture and Reagents. Primary human preadipocytes were isolated from healthy lean kidney donors aged 39 ± 3.3 y with a body mass index of 26.6 ± 0.9 (mean ± SEM). Primary preadipocytes also were isolated from five young (31 ± 5 y) and five old (71 ± 2 y) healthy male volunteers for SABG assay. The protocol was approved by the Mayo Clinic Foundation Institutional Review Board for Human Research. Informed consent was obtained from all human subjects. The preadipocyte isolation procedure and steps taken to ensure culture purity have been described previously (65). To induce senescence, preadipocytes were subjected to 10 Gy of cesium radiation. They were senescent by 20 d after irradiation, with little cell growth and more than 70% of cells exhibiting SABG positivity. Preadipocytes also were induced to become senescent by 27–30 serial passages, at which point cell proliferation was attenuated and more than 80% of cells exhibited SABG positivity. Preadipocytes were isolated from Brown Norway rats as previously described (66). HUVECs were purchased from ATCC. CYT387 (CAS 1056634-68-4) and ruxolitinib (INCB18424, CAS 941678-49-5) were purchased from ChemieTek. SABG Assay. Cellular SABG activity was assayed as previously described (21). In brief, primary preadipocytes or fat tissues were washed three times with PBS and then were fixed for 5 min in PBS containing 2% (vol/vol) formaldehyde (Sigma-Aldrich) and 0.25% glutaraldehyde (Sigma-Aldrich). Following fixation, cells or tissues were washed three times with PBS before being incubated in SABG activity solution (pH 6.0) at 37 °C for 16–18 h. The enzymatic reaction was stopped by washing cells or tissues three to five times with ice-cold PBS. Fluorescence microscopy (Nikon Eclipse Ti) was used for imaging. About 8–10 images were taken from random fields in each sample. DAPI (Life Technologies) was used to stain nuclei for cell counting. CM. CM were made by exposing cells to RMPI 1640 containing 1 mM sodium pyruvate, 2 mM glutamine, MEM (minimum essential medium) vitamins, MEM nonessential amino acids, and antibiotic (Life Technologies). Nonproliferating healthy and senescent preadipocytes were washed three times with PBS and then were cultured with CM for 24 h. For JAK inhibitor treatment, senescent preadipocytes were treated with JAK inhibitor or DMSO for 48 h, washed three times with PBS, and cultured with CM containing JAK inhibitor or DMSO for another 24 h. Multiplex Protein Analyses. Luminex xMAP technology was used to quantify cytokines, chemokines, and growth factors. The multiplexing analysis was performed using the Luminex 100 system (Luminex) by Eve Technologies Corp. Human and mouse multiplex kits were from Millipore. More than 80% of the targets were within the detectable range (signals from all samples are higher than the lowest standard). Undetectable targets were excluded from Tables S1 and S2. Real-Time PCR. RNA from tissues or cells was extracted using TRIzol (Life Technologies) and was reverse transcribed to cDNA using the M-MLV Reverse Transcriptase kit (Life Technologies) following the manufacturer’s instructions. Real-time PCR was performed using TaqMan fast advanced master mix (Life Technologies). The probes and primers were purchased from Life Technologies. TATA-binding protein was used as an internal control. siRNA Knockdown. Senescent cells were transfected with 30 nM siRNA using Lipofectamine RNAiMAX (Life Technologies) following the manufacturer’s instructions. The cells were collected 48 h after transfection. All siRNAs were obtained from Life Technologies. The siRNA sequences were as follows: siJAK1-a: sense 5′-GGC AUG CCG UAU CUC UCC Utt-3′; antisense 5′-AGG AGA GAU ACG GCA UGC Ctg-3′; siJAK1-b: sense 5′-GGU UCU AUU UCA CCA AUU Gtt-3′; antisense 5′- CAA UUG GUG AAA UAG AAC Ctc-3′; siJAK2-a: sense 5′- GGU GUA UCU UUA CCA UUC Ctt-3′; antisense 5′-GGA AUG GUA AAG AUA CAC Ctg-3′; siJAK2-b: sense 5′-GGA GUU UGU AAA AUU UGG Att-3′; antisense 5′-UCC AAA UUU UAC AAA CUC Ctg-3′. Two nontargeting negative control siRNAs (catalog nos. AM4635 and AM4615) also were purchased from Life Technologies. Immunostaining. Cells were fixed with 4% (vol/vol) paraformaldehyde for 10 min and were permeabilized using PBS containing 0.25% Triton X-100 for 10 min. After being washed with PBS, cells were blocked with PBS and Tween-20 (PBST) containing 1% BSA for 30 min. Cells were incubated with primary antibodies in 1% BSA in PBST overnight at 4 °C and then with the secondary fluorescent antibodies (Life Technologies) in the same buffer for 1 h in the dark. DAPI (Life Technologies) was used to stain nuclei for cell counting. Fluorescence microscopy (Nikon Eclipse Ti) was used for imaging. Paraffin rat fat sections were deparaffinized with Histoclear (National Diagnostics) and hydrated by ethanol gradient. Antigen was retrieved by

Xu et al.

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incubation in 0.01 M citrate buffer (pH 6.0) at 95 °C for 10 min. Slides were placed in blocking buffer (normal goat serum 1:60 in 0.1% BSA in PBS) for 60 min at room temperature. Samples were blocked further with Avidin/ Biotin (Vector Laboratories) for 15 min each at room temperature. Primary antibody was applied overnight at 4 °C in the blocking buffer. Then slides were incubated for 30 min with biotinylated, anti-rabbit secondary antibody (Vector Laboratories). Finally, Fluorescein Avidin DCS (Vector Laboratories) was applied for 20 min. Samples were mounted in VECTRASHIELD DAPI-containing medium (Vector Laboratories) and were imaged using a LSM 780 Zeiss confocal microscope under the ×100 objective. γH2AX antibody was purchased from Cell Signaling, and Ki-67 antibody was purchased from Abcam. Western Immunoanalysis. Cells or tissues were homogenized in cell lysis buffer (Cell Signaling) with protease inhibitors (Sigma). The lysate then was cleared by centrifugation (16,000 × g for 10 min at 4 °C). Total protein content was determined using Coomassie Plus reagents (Pierce). Proteins were loaded into each lane on a 4–20% gradient SDS/PAGE gel and transferred to immunoblot PVDF membranes (Bio-Rad). Signals were developed with HRPconjugated secondary antibodies and SuperSignal West Pico Chemiluminescent Substrate (Pierce). The p21 antibody was purchased from Santa Cruz. All other antibodies were purchased from Cell Signaling. Macrophage Migration Assay. The THP-1 cell line was purchased from the ATCC. Migration assays were performed using Transwell polycarbonate membrane inserts with a 5-μm pore diameter (Corning). Three hundred microliters of CM collected from cells or blank CM (without exposure to cells) were introduced into the bottom chambers of the plates in triplicate. THP-1 cells (105) resuspended in blank CM were plated into the inserts. THP-1 cells were allowed to migrate into the bottom chamber for 6 h at 37 °C. After 6 h, the number of cells that had migrated into the lower chamber was quantified using the CyQUANT cell proliferation assay kit (Life Technologies) according to the manufacturer’s instructions. The number of cells migrating into blank CM was considered as background and was subtracted from the experimental groups. Mice and Drug Treatments. Twenty-four-month-old C57BL/6 male mice were obtained from the National Institute on Aging and were maintained in a pathogen-free facility at 24 °C under a 12 h/12 h light/dark cycle with free access to food (standard mouse diet; Lab Diet 5053) and water. For drug treatment, ruxolitinib was dissolved in DMSO and then was ground and mixed with food. In addition to regular food, each mouse was fed a small amount of food (0.5 g) containing ruxolitinib (60 mg/kg body weight) or DMSO daily. During the 10 wk of treatment, all mice consumed the drug-containing food completely every day. The experimental procedure was approved by the Institutional Animal Care and Use Committee at Mayo Clinic. Measurements of Physical Function. Forelimb grip strength [N (newton)] was determined using a Grip Strength Meter from Columbus Instruments following a 3-d acclimation. Results were averaged from six trials. For the hanging test, mice were placed on a 2-mm-thick metal wire, which was placed 35 cm above a padded surface. Mice were allowed to grab the wire only with their forelimbs. The hanging time was normalized to the body weight as hanging duration in seconds × body weight in grams. The results were averaged from three trials for each mouse. Coordination was assessed using an accelerating RotaRod system (TSE Systems). Mice were trained on RotaRod at a speed of 4 rpm for 200 s for 3 d. On the test day, mice were placed onto the stationary RotaRod. The RotaRod then was started at 4 rpm and accelerated to 40 rpm over a 5-min interval. The speed when the mouse dropped from the RotaRod was recorded. Results were averaged from three trials. Comprehensive Laboratory Animal Monitoring System. In a subset of eight mice per group, the habitual ambulatory, rearing, and total activity, oxygen consumption (VO2), and carbon dioxide production (VCO2) of individual mice were monitored over a 24-h period (12 h light/12 h dark) using a Comprehensive Laboratory Animal Monitoring System (CLAMS) equipped with an Oxymax Open Circuit Calorimeter System (Columbus Instruments). Ambulatory, rearing, and total activity was summed and analyzed for light and dark periods under fed conditions. The VO2 and VCO2 values were used to calculate the respiratory exchange ratio (RER) and VO2. RER values were used to determine the basal metabolic rate (in kilocalories per kilogram per hour). Statistical Analysis. Analyses of differences between groups were performed by Student’s t test, one-way ANOVA, or repeated-measures ANOVA where appropriate. Values are presented as mean ± SEM with P < 0.05 considered to be significant.

PNAS | Published online November 2, 2015 | E6309

CELL BIOLOGY

Methods

ACKNOWLEDGMENTS. We thank Dr. Ann Oberg for statistical support and colleagues in the Robert and Arlene Kogod Center on Aging for comments and discussion. This work was supported by NIH Grants DK50456 (to

M.D.J.), AG041122 (to J.L.K.), and AG013925 (to J.L.K.). M.X. received a Glenn/American Federation for Aging Research Postdoctoral Fellowship for Translational Research on Aging.

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