Loss of Progranulin Expression Decreases NLRP3 Inflammasome-Mediated Inflammation and Enhances Bone Anabolism

Loss of Progranulin Expression Decreases NLRP3 Inflammasome-Mediated Inflammation and Enhances Bone Anabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Loss of Progranulin Expression Decreases NLRP3 Inflammasome-Mediated Inflammation and Enhances Bone Anabolism Robert Nissenson, Vikrant Piprode, Liping Wang, Gursimar Kohli, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9441627/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Progranulin (PGRN), a glycosylated protein, is expressed in most tissues, including bones, and its level is elevated in the serum and joints of individuals with inflammatory bone loss disorders such as rheumatoid arthritis (RA). Previously, using global and macrophage-specific Grn deletion mice, we demonstrated that loss of PGRN protects against age-related bone loss selectively in females, suggesting a sex-dependent role for macrophage-derived PGRN in skeletal homeostasis. Here, we investigated the role of PGRN in the regulation of macrophage-mediated inflammation and bone formation. Immune-phenotyping revealed that Grn −/− vs. WT mice exhibited higher percentages of Mac2 Hi subsets in both sexes. Transcriptomic analysis of Mac2 Hi vs. Mac2 Lo from WT mice showed reduced expressed expression of Nlrp3 and Il1β at baseline in both sexes. Further, Grn −/− vs. WT M-CSF-dependent macrophages (BMMs) revealed decreased expression of Nlrp3 and Il1β following LPS challenge in vivo in both sexes. Consistent with this, Grn −/− mice displayed markedly reduced IL-1β production in serum and paw joints, and attenuated bone erosion in the STA-induced RA model, indicating altered NLRP3 inflammasome signaling in Grn −/− mice. Notably, PGRN deficiency enhances the osteoanabolic capacity of macrophages: female Grn −/− BMMs potentiated osteogenic differentiation of mesenchymal stem cells, and in vivo , Grn −/− females exhibited higher trabecular bone formation in response to intermittent PTH. Collectively, PGRN deficiency in BMMs is negatively associated with Nlrp3 expression and IL-1β production and causes reduced inflammation and bone erosion in mice subjected to STA-induced RA. Furthermore, PGRN limits the bone-anabolic action of PTH in a female sex-specific manner. Biological sciences/Physiology/Bone Health sciences/Pathogenesis Progranulin macrophages inflammation NLRP3 inflammasomes IL-1β arthritis iPTH Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Progranulin (PGRN), a glycoprotein encoded by the Grn gene, is constitutively expressed in different tissues, including epithelia, particularly in skin, gastrointestinal tract, reproductive system, immune cells, specific neurons, microglia, and adipose tissue 1 , 2 . Intracellularly, PGRN is in lysosomes, where it regulates protein trafficking, functions as a molecular co-chaperone and helps acidification of lysosomes 3 , and as secretory protein, PGRN acts as a growth factor 4 – 7 .It binds to several cell surface receptors such as tumor necrosis factor-α receptor 1 and 2 8 , Sortilin Related VPS10 Domain Containing Receptor 2 9 , EGFR 10 , and Ephrin A2 11 , to mediate differential cellular functions. Consequently, PGRN plays a pivotal role in embryogenesis, inflammation, wound healing, and tumorigenesis 1 . In humans, a heterozygous loss-of-function (LOF) mutation in Grn causes Frontotemporal lobar dementia (FTLD/FTD), while homozygous LOF mutation leads to neural ceroid lipofuscinosis type 11 (NCL11), characterized by vision loss, dementia and epilepsy 12 . PGRN has been widely implicated in immune regulation, particularly in macrophage biology. It inhibits LPS-induced M1 macrophage polarization of RAW264.7 cells, bone marrow-derived macrophages (BMMs), and THP-1 cells 13 . Consistent with its anti-inflammatory effects, Grn −/− mice undergo spontaneous osteoarthritis and exhibit exacerbated inflamed joints in collagen-induced arthritis (CIA) model 14 , 15 . Moreover, PGRN enhances M2 macrophage polarization of BMMs and RAW264.7 as shown by a decrease in CD86 expression (M1 marker) and increase in CD206 expression (M2 marker) 16 . In vivo , recombinant PGRN increased CD206 + macrophages in unilateral ureteral obstruction (UUO) model 17 . Furthermore, Grn −/− mice were protected mice from endotoxic shock following LPS injection and showed reduced lung injury 18 . All these studies indicated an anti-inflammatory role of PGRN. Conversely, higher serum PGRN levels are reported in RA, OA and Type 2 Diabetes (T2D) individuals. In mice, PGRN mediates high fat diet-induced insulin resistance via IL-6 production in adipose tissue 19 . Similarly, in severe SLE individuals, higher serum PGRN levels positively correlate with higher serum IL-6 and TNFα 19 . Further, PGRN stimulates secretion of IL-8 by epithelium cells in Multiple Sclerosis (MS), which acts as chemotactic factor for neutrophils and monocytes 19 . Thus, PGRN is one of those molecules whose imbalance in levels brings out different outcomes depending on the cell type and the diseases being examined, also known as Progranulinopathies, which include autoimmune diseases, metabolic, musculoskeletal and cardiovascular diseases. The dual function of progranulin: as a pro- and an anti- inflammatory molecule, depending on the cellular context is elaborated in the review by Huang et al 19 . Previously, we demonstrated that global deletion of Grn protects female but not male mice from aging-induced bone loss. A similar phenotype was observed in Cx3Cr1 Cre ; Grn f/f female mice, suggesting a critical role of macrophage-derived PGRN in regulating bone homeostasis in female mice 20 . Macrophages impact bone health, indirectly via inflammation, and directly act as osteoclast precursors, and as osteomacs to enhance or inhibit bone formation functions of osteoblasts depending on the macrophage phenotype 21 . Additionally, PGRN protects from TNF-ɑ-induced inhibition of osteoblast differentiation and mineralization 22 , underscoring its dual role in bone remodeling. In FTD individuals with GRN mutations, neuroinflammation due to microglia activation produces inflammatory cytokines including IL-1β and IL-18. These two cytokines are products of NLRP3 inflammasome cascade 23 . NLRP3, the NOD-, LRR-, and pyrin domain-containing 3 inflammasome, is a well-known innate immune pathway and is activated by DAMPs and PAMPs. Consequently, caspase-1 is activated which produces bioactive IL-1β and IL-18 to induce inflammatory cell death called pyroptosis 24 . These observations suggest an association of PGRN with NLRP3 inflammasome cascade. In the present study we investigated the role of PGRN in influencing macrophage functions which ultimately impact bone health. We find that Grn −/− BMMs are less inflammatory than the PGRN-replete BMMs. Mechanistically, Grn −/− BMMs exhibit down-regulation of Nlrp3 and Il1β expression and show poor response to LPS-induced inflammation and M1 polarization. As a result, Grn −/− mice show significant alleviation of inflammation in response to serum transfer-induced arthritis. Moreover, Grn −/− female BMMs mice show higher bone anabolic activity in vitro and in response to PTH administration, indicating that PGRN limits the anabolic action of PTH therapy. Mechanistically, our bulk RNA-seq data shows downregulation of G i signaling in the Grn −/− osteoblasts, which in part, can contribute to enhanced bone formation via activation of Gs signaling in response to PTH. In summary, these results suggest that PGRN expression is associated with NLRP3-inflammasome-mediated inflammation and suppression of bone anabolic functions of macrophages. RESULTS Freshly isolated macrophages from Grn −/− are Mac2 hi subset with decreased inflammatory phenotype As reported, female mice with PGRN deficiency in macrophages ( Cx3Cr1 Cre ; Grn f/f mice) are protected from aging-induced bone loss 20 . Thus, we began characterizing the freshly isolated bone marrow macrophages from WT and Grn −/− mice of both sexes 25 . We found similar percentages of total macrophages in Grn −/− and WT mice in both sexes expressing CD11b + /CD45 + /F4/80 + /Ly6G + /Ly6C + . Interestingly, in female Grn −/− mice, ~ 72.35% of these macrophages were Mac2 high (Mac2 Hi , positive for Mac2) and only ~ 23.39% were Mac2 low (Mac2 Lo , negative for Mac2). Conversely, in female WT mice, ~ 16.18% were Mac2 Hi and ~ 77.2% were Mac2 Lo (Fig. 1 A and 1 B). Male Grn −/− mice exhibited ~ 94.27% Mac2 Hi and ~ 3.09% Mac2 Lo macrophages, and male WT showed ~ 25.39% of Mac2 Hi and ~ 69.4% Mac2 Lo (Fig. 1 B). Furthermore, NanoString-based transcriptomics on FACS-sorted Mac2 Hi subsets displayed enrichment of cell cycle genes likely indicating a higher proliferation rate. Contrarily, Mac2 Lo macrophages showed higher expression of inflammation-related genes, notably, Nlrp3 and Casp1 (Fig. 1 C, 1 D ) . As activation of the NLRP3 inflammasome pathway is sufficient to polarize macrophage towards inflammatory M1 type, we focused on understanding NLRP3 inflammasome signaling in the macrophages. As Grn −/− have higher % of Mac2 Hi subset, we assessed NLRP3 inflammasome pathway in M-CSF dependent bone marrow-derived macrophages (BMMs) from WT and Grn −/− mice. We found that female Grn −/− vs. WT BMMs displayed reduced expression of Nlrp3 , Il1ß , Tnfa and Il6 (Fig. 1 E ) . However, male BMMs showed decreased Il1ß with other genes remained unchanged (Fig. 1 F ) . Additionally, female WT BMMs treated with rm PGRN for 24 hrs greatly enhanced the expression of Nlrp3 and Il1ß genes but not in males (Fig. 1 G and 1 H ) . These findings suggest an upstream regulatory role of PGRN in the expression of Nlrp3 pathway genes in female mice. Grn −/− mice exhibit a suppressed inflammatory response to LPS administration Our in vitro results suggested that macrophages from Grn −/− mice have lower Nlrp3 expression and possibly reduced NLRP3 inflammasome signaling. To corroborate our in vitro findings, we challenged female and male WT and Grn −/− mice with LPS (15 mg/kg body wt.) (Fig. 2 A). BMMs were prepared and subjected to NanoString-based transcriptome profiling. We observed that female Grn −/− BMMs showed differentially expressed genes with more than > 50-fold increase in the expression of Ccl2 and Ccl7 (Fig. 2 B), which are macrophage chemotactic proteins that also play a role in macrophage polarization. Of interest, Grn −/− vs. WT BMM showed reduced expression of Nlrp3 in response to LPS, which was further validated by qPCR (Fig. 2 C). Given that IL-1β production is largely NLRP3 inflammasome dependent, we indeed found that the serum IL-1β was lower in the LPS challanged Grn −/− vs.WT counterparts (Fig. 2 D ) . Reportedly, NLRP3 inflammasome signaling is involved in M1-type macrophage polarization. We found that LPS challenged female Grn −/− vs. WT BMMs displayed down-regulation of M1-type genes including iNOS, Tnfa, Fpr2, Cxcl10 , and Il6 (Fig. 2 E). Similarly, a reduced Nlrp3 expression and less serum IL-1β levels in the Grn −/− vs. WT was observed male mice ( Fig. 2 F and G ). Taken together, these results suggest that PGRN expression positively correlates with activation of the LPS-induced NLRP3 inflammasome signaling cascade, which in turn is required for M1-type macrophage polarization. Grn −/− mice show delayed onset of inflammation in response to STA-induced arthritis. We investigated the functional relevance of the association of PGRN with NLRP3-inflammasome dependent IL-1β production in the serum-transfer (STA)- induced rheumatoid arthritis (RA) mouse model 26 , 27 . Accordingly, Grn −/− and WT mice with KBxN or control serum, and arthritis score and paw thickness was measured from until day 5 (Fig. 3 A, B). Both male and female WT mice injected with KBxN serum showed robust inflammation at each time point until day 5 as observed by an increase in mean arthritis score (Fig. 3 C-F ) and paw thickness (Fig. 3 G and 3 H). Strikingly, both Grn −/− mice showed significantly lower arthritis scores and paw thickness compared to sex-matched WT counterparts (Fig. 3 C-H). To understand the cellular mechanism, freshly isolated bone marrow macrophages were immune-phenotyped on day 5. It was observed that the relative percentages of Mac2 Hi and Mac2 Lo subsets in both the sexes of WT and Grn −/− genotypes remained unchanged, irrespective of whether the mice received control or KBxN serum (Fig. 3 I and J ). Furthermore, we found that the levels of IL-1β were significantly higher in serum and ankle joint lysates from the WT vs. Grn −/− arthritic mice (Fig. 3 K and L ) and in both sexes. Our data provided important information that in the absence of PGRN both Nlrp3 gene expression and IL-1β cytokine levels are downregulated consistent with the attenuation of inflammation in STA model of RA in the Grn −/− mice. Thus, PGRN likely promotes inflammation via promoting the NLRP3 inflammasome cascade in WT mice subjected to STA induced RA. Grn −/− mice show mitigated inflammation during later stages of STA-induced arthritis and attenuated bone erosion In STA model of RA, inflammation starts to resolve gradually after day 5–7 and bone erosion is detectable by day 20 26, 28 . Therefore, we evaluated the dynamics of inflammation at later stages of RA and bone phenotype as the RA progresses in both the WT and Grn −/− mice of both sexes (Fig. 4 A). The Grn −/− vs. WT mice showed mitigation of inflammation throughout the progression of RA (Fig. 4 B and C) , evident by a significantly reduction in mean arthritic score and paw thickness (Fig. 4 D-G). Furthermore, on day 20, WT arthritic mice showed bone erosion around articular surfaces at the ankle joints, as assessed by µCT (Fig. 4 H). Strikingly, arthritic Grn −/− mice showed visible protection of erosive bone loss as compared to WT arthritic counterparts (Fig. 4 H ). In female Grn −/− mice, we did see a similar pattern (Fig. 4 H). Taken together, these data suggest that PGRN deficiency in mice reduces the severity of erosive bone loss in STA-induced RA. Grn −/− BMMs are highly efferocytic and enhance osteogenic differentiation of bone marrow stromal cells In the current study, we found that Grn −/− BMMs are mostly galectin3 + (Mac2 Hi ). Since galectin3 is reported to enhance the efferocytic potential of human monocytes in phagocytosing apoptotic neutrophils 29 , we assessed the efferocytic potential of PGRN-deficient BMMs. This was achieved by co-culturing labelled apoptotic OCY545 cells with BMMs from either WT and Grn −/− mice for 24 hrs and later counting the labelled BMMs (a read out of efferocytosis) using fluorescence microscopy. We found that Grn −/− BMMs showed a higher percentage of labeled cells (red) due to increased efferocytosis of apoptotic OCY545 as compared to WT BMMs at 2 months and 7 months of age, and in both the sexes (Fig. 5 A-C). These results suggest that PGRN limits macrophage efferocytic potential, possibly via downregulation of Mac2 expression. It is well established that osteal macrophages are critical to bone formation via different mechanisms, including depletion of apoptotic osteoblasts and by the recruitment of osteoblast precursors 30 . Also, the osteogenic differentiation of bone marrow stem cells is influenced by osteal macrophages and further influenced by macrophage phenotype, with M1 being inhibitory and M2 with stimulatory actions 31 . We assessed the potential of PGRN deficient BMMs to regulate bone marrow stromal cell osteogenic differentiation. Female WT BMSC co-cultured with WT BMMs displayed enhanced ALP + area and Von Kossa + area as compared to the control WT BMSC alone cultures (Fig. 5 D ) . However, intriguingly, female Grn −/− BMMs co-cultured with WT-BMMs exhibited an increased ALP + and Von Kossa + areas compared with either the WT BMM + WT BMSC co-culture or WT BMSC alone cultures (Fig. 5 D, E and F) . This suggests that BMMs from female Grn −/− mice have greater pro-osteogenic activity than BMMs from female WT mice. In the co-culture experiments using male mice, WT BMMs did enhance the osteogenic differentiation of BMSCs as compared to BMSC cultures with no BMMs added to them, as assessed by the increase in the percent ALP + and Von Kossa + areas (Fig. 5 D, H and I) . However, BMMs from male Grn −/− mice did not display enhanced pro-osteogenic activity compared to BMMs from male WT mice. We further confirmed these observations using qPCR-based relative gene expression of osteogenic marker genes, including Alp, Runx2, Col1, and Ocn. Gene expression studies of the co-cultures clearly showed that female Grn −/− BMM have higher pro-osteogenic activity than the WT-BMMs (Fig. 5 J), while male Grn −/− BMMs did not further enhance the expression of osteogenic genes as compared to BMM from WT male counterparts (Fig. 5 K). Taken together, these results suggest that the PGRN deficient BMMs derived from female Grn −/− mice have greater pro-osteogenic supporting potential than WT BMMs or male Grn −/− BMMs. Female Grn −/− mice display greater bone anabolic action of iPTH in vivo In our current study, we observed that BMMs derived from female Grn −/− mice showed greater pro-osteogenic function in driving the osteogenic differentiation of BMSCs. Also, it has been reported that macrophages are essential for bone anabolic actions of iPTH therapy in mice 32 . Thus, we evaluated whether PGRN-deficient macrophages are different in responses to iPTH therapy-induced bone anabolic actions in vivo and if sex-differences are observed. well. To achieve this, we s.c. injected both WT and Grn −/− mice with PTH (80 µg/kg/day) for 5 days/ week for 4 weeks. The L 5 vertebrae were subjected to bone phenotyping using mCT after 4 weeks of PTH treatment. We observed that administration of PTH induced bone formation in both male and female WT mice and Grn −/− mice as shown by a significant increase in BV/TV, Tb.Th., and Tb.N. and a decrease in Tb.Sp. (Fig. 6 A-H). Notably, PTH-injected Grn −/− female mice ( Grn −/− +PTH) had an increased anabolic response to iPTH compared to their female WT counterparts injected with PTH (WT + PTH), as shown by a higher BV/TV, Tb.Th., and Tb.N. with a significant decrease in Tb.Sp. (Fig. 6 A-D). However, PTH-injected Grn −/− male mice ( Grn −/− +PTH) showed similar parameters of trabecular bone to male WT counterparts injected with PTH (WT + PTH). These data are consistent with our in vitro findings of the pro-osteogenic function of PGRN-deficient macrophages. Furthermore, immunostaining of F4/80 + osteal macrophages on the femur bone surface of the WT and Grn −/− mice did not show any difference in osteal macrophage number in any of the groups or between the sexes ( Suppl. Figure 1 ). Moreover, BMMs derived PTH injected mice, both female and male exhibited a pattern of increased expression of efferocytic macrophage marker genes, including Arg1 , Il10 , Cd36 and SRA , irrespective of the sex and genotype. Transcriptomic profiling of female Grn −/− bone marrow stromal cells (BMSCs) reveal downregulation of inhibitory Gi signaling pathway In the present study, we found that female Grn −/− BMMs have greater potential of promoting osteogenic differentiation of BMSCs in vitro , and in vivo PTH stimulated higher bone anabolic effects in female Grn −/− mice. Thus, we performed RNAseq on the BMSCs after 21 days of osteogenic differentiation from both WT and Grn −/− to understand any intrinsic differences. We found that Grn −/− BMSCs showed > 5-fold increase in expression of genes of cell cycle progression, and > 3-fold decrease in the expression of genes that belong to inhibitory Gi signaling. Further Reactome analysis indeed showed upregulation of cell cycle genes and down-regulation of genes involved in Gi signaling in the Grn −/− BMSCs vs. WT-BMSCs (Fig. 7 A, B). We know from our previous work that inhibition of Gi signaling with pertussis toxin, using genetic mouse models, enhances bone formation in aging females, and accelerates bone anabolic action of PTH only in female mice 33 , 34 . The resulting active Gsα signaling pathway, which is a known PTH target, mediates the bone formation function of osteoblasts. DISCUSSION Available reports indicate PGRN has dual action, acting as a pro- or anti-inflammatory molecule depending on the cellular context 35 . Full-length PGRN can be digested by proteases, both intracellularly or extracellularly, into different granulins including, G, F, B, A, C, D, and E, which are known to play pro-inflammatory roles 12 . Reportedly, PGRN is anti-inflammatory in mouse models of osteoarthritis (OA) and collagen-induced arthritis (CIA) and is attributed to its potential to block the TNF-ɑ pathways by blocking its receptors (TNFRs) 14 , 15 . Similarly, Attstrin, PGRN analog, is anti-inflammatory 8 . In addition to its anti-inflammatory role, PGRN is essential for efficient osteoclast differentiation as the Grn −/− OC precursors are resistant to RANKL- induced osteoclast formation in vitro 34 . Additionally, PGRN protects from the inhibitory action of TNF-α on osteoblasts differentiation 22 . Previously, we reported that female Grn −/− are resistant to aging-associated bone loss. This is partly attributed to higher bone formation rate and reduced osteoclastic bone resorption. However, male Grn −/− mice do not exhibit such an aging phenotype and continue to lose bone despite reduced osteoclastic bone resorption. However, the mechanism(s) underlying the female-sex specific aging-associated bone protective role of PGRN is not clear. The female-specific bone protective role of PGRN was also seen in Cx3Cr1 Cre ; Grn f/f mice, suggesting that the negative role of PGRN on bone mass results from production of the protein by macrophage lineage cells. Thus, in the present study we investigated the role of PGRN in mediating the effects of macrophages on bone homeostasis. Firstly, to understand the role of PGRN in macrophage biology, we characterized freshly isolated bone marrow macrophages 25 . We found that Grn −/− mice have a higher percentage of Mac2 Hi macrophages in both male and female mice, suggesting that PGRN may have a role in the regulation of Mac2 expression. Mac2, also known as galectin3, is upregulated in microglia of patients with haploinsufficiency due to loss-of-function mutation in Grn genes. Galectin3 has been reported to be essential for human macrophage invasion and for suppressing pro-inflammatory cytokine production 36 . Interestingly, our transcriptomics data indeed revealed that Mac2 Hi macrophages express low levels of inflammation-related genes, and notably, we found that Nlrp3 was downregulated in these PGRN-deficient macrophages. As NLRP3 inflammasome signaling is involved in the maturation and secretion of IL-1β, we found that the serum IL-1β levels and Nlrp3 expression in the M-CSF dependent BMMs were lower in the Grn −/− mice as compared to the WT in response to LPS challenge, thus suggesting that PGRN expression is positively correlated with NLRP3 inflammasome signaling pathway gene expression. To understand the disease relevance of these findings, we employed a serum transfer-induced rheumatoid arthritis (RA) model. This model of RA is principally dependent on IL-1β-mediated inflammation and bone loss. The results clearly indicated that PGRN presence is required for effective NLRP3 mediated-IL-1β-induced inflammation as the Grn −/− showed signs of alleviated inflammation in the paw in both the sexes and that the mice showed reduced severity of bone erosion. The reduced serum and paw joints levels of IL-1β in Grn −/− further suggests that PGRN regulates NLRP3 inflammasome signaling leading to IL-1β-mediated inflammation, arthritis and the subsequent bone erosion in WT mice. In the present study, M-CSF dependent BMMs from both male and female Grn −/− mice demonstrated enhanced efferocytotic activity in vitro . Increased efferocytotic activity of macrophages has been reported to be associated with increased bone formation in vivo 30 , which is due to clearance of apoptotic mature osteoblasts and recruiting new osteoblast precursors at the site of bone formation. These efferocytic macrophages are reported to be F4/80 + osteomacs present on the bone surface. Further, Cho et al. in 2014 reported that these F4/80 + osteomacs are critical for the anabolic action of intermittent parathyroid hormone therapy (iPTH) 32 . Interestingly, we found that BMMs from female Grn −/− co-cultured with bone marrow stromal cells (BMSCs) from WT mice showed greater osteogenic differentiation potential than the WT BMMs or BMMs alone However, BMMs from male Grn −/− co-cultured with bone marrow stromal cells (BMSCs) did not show enhanced osteogenic differentiation from WT counterparts. Further, in vivo , we found that the female Grn −/− mice showed enhanced anabolic action of iPTH in L5 vertebrae as compared to their WT counterparts. These results further highlight the importance of macrophage efferocytotic function as an essential process of iPTH therapy. However, for reasons unknown, in our study the enhanced anabolic effect of iPTH was limited only to female Grn −/− despite increased efferocytic function in both the sexes in vitro . Further, the transcriptomics and reactome analysis of BMSCs from Grn −/− revealed downregulation of genes involved in the Gi signaling pathway. Intriguingly, Gi signaling has been well documented to hamper bone formation and to limit the anabolic action of iPTH therapy in female mice 33 , 34 . One such Gi-GPCR is Htr1b which was down-regulated in the female Grn −/− BMSCs. It is well-documented that gut-derived serotonin binds to Htr1b on osteoblasts and inhibits bone formation via decreasing cAMP response element-binding protein (CREB) function, a key transcription factor that promotes osteoblast proliferation and differentiation 37 . Thus, an increased pro-osteogenic and efferocytic functions of macrophages together with inhibition of Gi signaling in Grn −/− females can partly explain the enhanced bone anabolic action of iPTH therapy. This could possibly explain our previous finding that only female Grn −/− mice are resistant to aging-associated bone loss 34 . However, further mechanistic studies are required to understand the sexual dimorphic actions of PGRN on aging-induced bone loss. In summary, deficiency of PGRN expression in mouse macrophages downregulates Nlrp3 and Il1β expression to alter NLRP3 signaling cascade which confers macrophage with less inflammatory phenotype. Consequently, the severity of bone erosion is reduced in Grn −/− serum transfer-induced RA model in mice. Furthermore, these PGRN-deficient macrophages exhibit enhanced osteogenic potential that can contribute to greater anabolic action of PTH in Grn −/− female mice and protect these mice from aging-associated bone loss. However, molecular mechanisms governing progranulin-mediated activation of the NLRP3 inflammasome and its bone anti-anabolic effects are unclear and will be of great interest for further studies. MATERIALS AND METHODS Animals All animal studies were approved by and performed in accordance with the Institutional Animal Care and Use Committees at the San Francisco VA Medical Center and the University of California, San Francisco (UCSF). We bred heterozygous Grn −/+ mice in C57BL/6 background, generously provided by Dr. Robert V. Farese at UCSF, to generate Grn −/− and WT littermate (used as controls). KRN mice, provided by Dr. Clifford Lowell at UCSF, were bred with NOD mice (The Jackson Laboratory, Bar Harbor, ME) to generate K/BxN mice. Grn −/− and WT mice of both sexes and various ages were used in different experiments. Isolation of mouse bone marrow-derived macrophage and culture of M-CSF dependent macrophages (BMMs) Freshly isolated bone marrow macrophages were immune-phenotyped (flow cytometry section) or cultured with M-CSF to generate M-CSF-dependent macrophages (BMMs) 25 . Briefly, mice were euthanized and hindlimb bones were excised, demuscled, and the epiphyseal ends were cut open, and the marrow was flushed with RPMI-1640 (Gibco) using a syringe with a needle size of 26 1/2 gauge. Upon RBCs lysis using Lysis Buffer (Cat#00-4333-57, eBioscience), cells were washed and resuspended in RPMI-1640 growth medium containing 10% FBS, 1% penicillin-Streptomycin, and 0.1% Fungizone, and 20 ng/mL macrophage-colony stimulating factor (M-CSF; Cat# 416-ML-050/CF, R&D Systems) for 24 hours. Next day, the non-adherent fraction is collected and cultured in RPMI-1640 growth medium with M-CSF (20 ng/mL) for 6 days to generate macrophages (BMMs). To understand the effect of PGRN deficiency or action of exogenous PGRN on the expression of the proinflammatory genes, BMMs were prepared from 12-weeks-old Grn −/− and their littermate WT. The BMMs were treated for 24 hrs with 500 ng/mL of recombinant mouse PGRN (Cat# AG-40A-0189Y-C010, AdipoGene Life Sciences). Bone marrow stromal cells (BMSCs) isolation and co-culture with BMMs Bone marrow macrophages (BMMs) were prepared from 10-month-old Grn −/− or WT mice as described above. On the day before the co-culture assay, male and female C57BL/6 mice of 10 months age were used for isolating bone marrow stromal cells (BMSCs). In brief, total bone marrow was flushed out and BMSCs were enriched with MACS technology by depleting the mature hematopoietic lineage cells with CD11b + MACS microbeads (Cat#130-126-725, Miltenyi Biotec). The enriched BMSCs were plated in a 6-well plate at a density of 3 x 10 6 cells/well for female cell BMSCs and 2.6 x 10 6 cells /well for male BMSCs (day 0). On day 1, bone marrow macrophages (BMMs) were enzymatically freed and then seeded into the culture wells containing BMSCs in a ratio of 1:7 (BMMs/BMSCs). The cultures were maintained undisturbed for 5 days in a 5% CO 2 maintained at 37°C. culture medium was removed along with all non-adherent cells and replaced with fresh alpha MEM with 50 µg/ml ascorbic acid and 3 mM β-glycerophosphate to initiate osteogenic differentiation. The culture medium was replaced every three days. At day 21, alkaline phosphatase and Von Kossa stainings were performed using ALP staining kit (Cat# ab284936, Abcam) and Silver Nitrate staining methods, respectively. Stained areas were quantified using NIH Image J software. RNA isolation, cDNA preparation and qPCR Total RNA was isolated from the cells using TRI reagent-based phenol-chloroform isolation followed by purification with RNeasy mini kit (Cat# 74104, Qiagen), according to the manufacturer’s instructions. The isolated Total RNA was used to prepare cDNA using TaqMan™ Reverse Transcription Reagents (Cat# N8080234, Thermo-Fisher) according to the manufacturer's instructions. qPCR was performed using 10 ng of cDNA using SYBR™ Green Universal Master Mix and 100 nM of forward and reverse primer pairs for each gene, designed using Primer Bank. Gapdh was used as an endogenous control gene, and the relative expression of the gene was calculated using 2 − dCT . Primers used for all genes are listed in Table 1 . Table 1 List of all qPCR primers Primer Forward Primer (5’-3’) Reverse Primer (5’-3’) Nlrp3 AGA AGA GAC CAC GGC AGA AG CCT TGG ACC AGG TTC AGT GT Il1β GTG CAA GTG TCT GAA GCA GC CAA AGG TTT GGA AGC AGC CC Tnfα GCC TCC CTC TCA TCA GTT CTA GGC AGC CTT GTC CCT TG Il6 ATC CAG TTG CCT TCT TGG GAC TGA TAA GCC TCC GAC TTG TGA AGT GGT iNOS GAG ACA GGG AAG TCT GAA GCA C CCA GCA GTA GTT GCT CCT CTT C Fpr2 GAG CCT GGC TAG GAA GGT G TGC TGA AAC CAA TAA GGA ACC TG Cxcl10 CCA AGT GCT GCC GTC ATT TTC GGC TCG CAG GGA TGA TTT CAA Bglap CTG ACC TCA CAG ATG CCA AG GTA GCG CCG GAG TCT GTT C Col1 GCG AAG GCA ACA GTC GCT CTT GGT GGT TTT GTA TTC GAT GAC Runx2 CGA GAC CAA CCG AGT CAT TT ACG CCA TAG TCC CTC CTT TT Arg-1 TGT CCC TAA TGA CAG CTC CTT GCA TCC ACC CAA ATG ACA CAT Il10 CTG GAC AAC ATA CTG CTA ACC G GGG CAT CAC TTC TAC CAG GTA A Cd36 ATT AAT GGC ACA GAC GCA GC GCA TTG GCT GGA AGA ACA AA SRA GTC GGG ATC TCC TGG ACC TA ATC CCA GCG ATC ATC ACA GA Gapdh TGC ACC ACC AAC TGC TTA G GGA TGC AGG GAT GAT GTT C Flow cytometry Freshly isolated adult mouse bone marrow macrophages were characterized as described previously 25 . In brief, freshly isolated bone marrow cells after RBS lysis were incubated with TruStain FcX to block the Fc receptors and cells were stained with fluorescence tagged antibodies as listed in Table 2 . BD compensation beads were used for setting up compensation. Samples were acquired using BD FACSAria™ FUSION and populations were sequentially gated via FMOs. Table 2 List of antibodies used for flow cytometry Antibody Catalog Company TruStain FcX (Clone 93) 101320 Biolegend Pacific Blue anti-mouse CD45 (clone 30-F11) 103126 Biolegend Allophycocyanin (APC) F4/80 (clone BM8) 123116 Biolegend APC-Cy7 rat anti-CD11b (clone M1/70 557657 Biolegend PE/Dazzle 594 Ly-6G (clone1A8) 127648 Biolegend Spark UV™ 387 anti-mouse Ly-6C Antibody (clone HK1.4) 128059 Biolegend Alexa Fluor 488 Mac2 (clone M3/38) 125410 Biolegend NanoString nCounter 7-months-old male and female C57BL/6 mice were used to evaluate the differential transcriptome of Mac2 Lo vs Mac2 Hi in freshly isolated macrophages population. In brief, freshly isolated mouse macrophages were enriched with CD11b + MACS microbeads, incubated with Fc block, and then stained for Mac2 antibody (Table 2 ) to sort Mac2 + cells with high (Mac2 Hi ) and low Mac2 (Mac2 Lo ) with a BD FACSAria™ FUSION at the SF VAMC FACS core. RNA was extracted from the sorted Mac2 Lo and Mac2 Hi cells with an Invitrogen PureLink RNA Micro Scale kit (Waltham, MA). Similarly, RNA from M-CSF dependent BMMs harvested from LPS-challenged WT and Grn −/− were subjected to NanoString nCounter system (Seattle, WA) using a nCounter® Myeloid Innate Immunity Panel. LPS challenge experiment Male and female Grn −/− and WT littermates of 6-9-months-age were i.p. injected with 15 mg/kg Lipopolysaccharide (LPS) (Cat# L4130, Escherichia coli O111:B4, Sigma, MO). After 6 hrs, the mice are euthanized, bone marrow was collected and M-CSF dependent BMMs were prepared. Subsequently, BMMs were subjected to NanoString-based gene expression analysis. Blood serum was also collected for cytokine level measurement. Induction of serum-transfer-induced (STA) rheumatoid arthritis We bred the KRN mice with NOD mice to obtain the K/BxN mice. The blood is collected from K/BxN mice at the age of 8–10 weeks old and allowed to clot at RT for 15 min and then centrifuged at 2000 x g for 10 min at 4°C. The KBxN serum is then stored in -80 until use. We used the serum from C57BL/6 mice of the same age as control serum. The experiment involved injection of 100 µL of K/BxN serum or control serum in 2- months-old WT and Grn −/− male and female mice. Two injections were done one on day 0 and on day 1 i.p. As a measure of inflammation, arthritis score and paw thickness were measured on each day until day 5 post first injection for the inflammatory phase study. For the effector phase study, the inflammation was monitored every three days until day 15 26, 28 . After euthanizing the mice, serum was collected, one ankle joint from each mouse was snap frozen for ELISA. The femurs were used to characterize the BMMs using flow cytometry. Measurement of arthritis score and paw thickness To measure inflammation in the RA mice, we used the arthritis scoring method as previously described 26 , 28 . For each of the four limbs (maximum four points per limb, up to a combined total of 16), score points (1–4) according to the presence of the feature with the greatest point value.1 point if there is only redness of the bottom of the footpad; 2 points if there is visible thickening of the paw, 3 points if the swelling of the ankle is sufficient to make the ankle equal to or greater in width than the mid footpad, 4 points if there is swelling of at least one digit. The paw thickness was measured using digital calipers at the ankle joint (malleoli) and is presented in millimeters (mm). Intermittent Parathyroid hormone (iPTH) administration 4-months-old WT and Grn −/− mice of both the sexes were injected s.c. with recombinant human PTH 1–34 (Bachem Inc., CA) (dissolved in 10 mM acetic acid in PBS with 2% heat inactivated C57BL/6 serum) at a dose of 80 µg/kg body weight for five consecutive days per week, for 4 weeks. Control animals were treated with the same volume of vehicle – 10% acetic acid PBS in PBS containing 2% heat-inactivated C57BL/6 serum. At the end of the experiment, mice were euthanized and L5 vertebrae and hind limbs were fixed in 4% PFA and then subjected to micro-CT (µCT) analysis for bone phenotyping. Micro-computed Tomography Hind limb paws with intact ankle joint from the RA mice and L5 vertebra from PTH-treated mice were fixed in 4% PFA for 48 hours at 4°C and then stored in 70% ethanol before being assessed using µCT scan and histomorphometry. Ankle joints were scanned using a Scanco VivaCT-50 µCT system (Scanco Medical, Brüttisellen, Switzerland) with an X-ray energy of 55 kV, a voxel size of 10.0 µm, and an integration time of 500 ms. We analyzed the 3D images of the whole ankle joint and visually assessed the bone erosion. For the PTH study, the trabecular region of interest (ROI) within L 5 was assessed. The ROI was defined as a cylindrical volume of 0.5 mm² cross-sectional area and trabecular parameters were evaluated. Serum and paw joint IL-1β measurement To assess the levels of cytokines in the serum in LPS-injected animals, we used a flow cytometry-bead based immunoassay using LegendPlex™ Mouse M1 Macrophage Panel (8-plex) (Cat#740848, Biolegend), according to the kit instructions. The concentration of each cytokine is subsequently determined by referencing a standard curve generated concurrently within the same assay using online LEGENDplex™ Data Analysis Software by applying a 5-parameter curve fitting algorithm. For serum-transfer induced arthritis studies, serum and paw-joint lysate IL-1β was measured using Mouse IL-1 beta/IL-1F2 DuoSet ELISA kit (Cat# DY401-05) following manufacturer’s instructions. Immunofluorescence Tibiae were collected from PTH or vehicle treated WT and Grn −/− mice and fixed in 4% PFA followed by demineralization. 5 µm thick sections were made using cryotome and subjected to IHC. Briefly, the sections were blocked with 5% donkey serum in PBS and then incubated with primary antibody against F4/80 (Cat# MF48000) overnight at 4 ο C in a humidified chamber. Next day, the sections are washed and incubated with Goat anti-rat Alex Flour™ 555 (Cat# A-21434). The slides were analyzed at 20X magnifications using the microscope BZ-X800 (Keyence). RNAseq BMSCs from WT and Grn −/− female mice (n = 3/genyotype) underwent osteogenic differentiation as described before. At day 21, RNA was isolated and RNAseq analysis was performed by Novogene using NovaSeq PE150 platform. Pathway analysis was carried out using the Reactome database and differentially regulated genes were assessed using threshold is normally set as: p adj < 0.05. Statistical analysis Statistical analysis was carried out using Graph pad Prism 10 software (version 10.6.1). For comparison of two groups, we used an unpaired student’s t-test. For experiments with more than 2 groups, we used one-way ANOVA with post-hoc Bonferroni’s correction and for more than 2 parameters, we used Two-way ANOVA. Each graph presented is a mean ± SEM. Declarations CONFLICT OF INTERESTS All authors declare that they have no conflicts of interest. CONTRIBUTIONS VP, LW, MN, GM and RAN, designed and planned the study. VP, LW, GK, PN, YW and CW performed all the experiments. VP and LW analyzed all data and performed statistical analysis. VP wrote the first draft which was later corrected by all the co-authors and approved the final version. ACKNOWLEDGEMENTS The authors would like to thank San Francisco VA Medical Center (SF VAMC) Bone Core for its technical assistance. This work was supported by the Veterans Affairs Merit Review Program (5I0BX003213 to R.A.N.), NIH P30 (2P30AR075055). The work was previously presented in parts as a poster at the American Society for Bone and Mineral Research Annual Meeting 2024 (Toronto, CA) and 2025 (Seattle, USA). DATA AVAILABILITY All datasets generated during and/or analyzed during the current study are presented in the article. Any additional raw files of transcriptomics are available from the corresponding author on reasonable request. References Bateman A, Bennett HP. The granulin gene family: from cancer to dementia. Bioessays. 2009;31(11):1245–54. doi: 10.1002/bies.200900086 . Review. Schmid A et al. Role of progranulin in adipose tissue innate immunity. Cytokine. 2020;125:154796. doi: 10.1016/j.cyto.2019.154796 . Epub 2019 Aug 24. Almeida S, Zhou L, Gao FB. 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Cell. 2008;135(5):825–837. doi: 10.1016/j.cell . 2008.09.059 Additional Declarations There is no conflict of interest Supplementary Files SupplementaryFig.docx Supplementary Figure 1 Cite Share Download PDF Status: Under Review Version 1 posted Reviewer # 2 agreed at journal 06 May, 2026 Reviewer # 1 agreed at journal 02 May, 2026 Reviewers invited by journal 27 Apr, 2026 Submission checks completed at journal 16 Apr, 2026 Editor assigned by journal 16 Apr, 2026 First submitted to journal 16 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9441627","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":630687210,"identity":"0df34554-bae9-46ea-a11c-0028851902fe","order_by":0,"name":"Robert 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\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice are Mac2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eHi\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e subset with decreased inflammatory phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Representative data from the flow cytometric analysis of the live bone marrow macrophages (CD45\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003ecells) from 7-months-old female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand littermate WT mice. The macrophages consisted of Mac2\u003csup\u003eLo\u003c/sup\u003e and Mac2\u003csup\u003eHi \u003c/sup\u003esubsets. The gates were determined through fluorescence minus one (FMO) controls. \u003cstrong\u003eB,\u003c/strong\u003e Distribution of Mac2\u003csup\u003eLo\u003c/sup\u003e and Mac2\u003csup\u003eHi\u003c/sup\u003e macrophage subsets (% of total analyzed cells) in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e- \u003c/sup\u003eand WT bone marrow from both the sexes. These subsets were sorted from WT mice and subjected to NanoString-based transcriptomics. \u003cstrong\u003eC\u003c/strong\u003e, Heatmap showing differential expression of genes from n=3 mice/ group (n1, n2, n3). \u003cstrong\u003eD\u003c/strong\u003e, normalized expression of \u003cem\u003eNlrp3\u003c/em\u003e in both females and males BMMs. The intrinsic expression of inflammatory genes including \u003cem\u003eNlrp3\u003c/em\u003e, \u003cem\u003eIl1β\u003c/em\u003e, \u003cem\u003eTnfα\u003c/em\u003e, and \u003cem\u003eIl6 \u003c/em\u003ewas assessed by qPCR in WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e. M-CSF dependent bone marrow-derived macrophages (BMMs) in \u003cstrong\u003eE\u003c/strong\u003e, female and \u003cstrong\u003eF\u003c/strong\u003e, male cohorts. WT BMMs from both sexes were treated with rPGRN at 500 ng/mL for 24 hrs and expression of these inflammation-related genes was assessed by qPCR. Data in \u003cstrong\u003eB\u003c/strong\u003e are presented as mean ± SEM (n=4 for each genotype in female cohort; and n=6 WT and n=4 \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e in male cohort), and was analyzed using Two-way ANOVA using uncorrected fisher LSD correction. Data in \u003cstrong\u003eD-H\u003c/strong\u003e, are presented as mean ± SEM of n=3 for each genotype in both the sexes and were analyzed using non-parametric t-test.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/6e3cbdbe5413e82abab37eef.jpg"},{"id":108805381,"identity":"c664c9ae-074d-44ba-8098-6e9b26a8d868","added_by":"auto","created_at":"2026-05-08 15:25:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice display attenuated LPS-induced NLRP3-inflammasome associated inflammatory responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Female and male WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice of 6-8 -months-old were i.p. administered with LPS (15 mg/kg body wt). After 6 hrs, M-CSF-denependent macrophages were preparaed and total RNA was isolated and subjected to Nanostring-based transcriptomics which was analyzed and a \u003cstrong\u003eB\u003c/strong\u003e, volcano plot showing the expression profiles of myeloid innate immune genes in red (up-regulated) and blue dots (down-regulated), assessed by Nanostring in the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-LPS vs. WT-LPS female mice. Bar graphs showing \u003cstrong\u003eC\u003c/strong\u003e, \u003cem\u003eNlrp3\u003c/em\u003e expression as assessed by qPCR, and \u003cstrong\u003eD\u003c/strong\u003e, serum IL-1β was evaluated using LEGENDplex\u003csup\u003eTM\u003c/sup\u003e array. \u003cstrong\u003eE\u003c/strong\u003e, M1 macrophage-related genes expression in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-\u003c/em\u003eLPS vs. WT-LPS. \u003cstrong\u003eF\u003c/strong\u003e and \u003cstrong\u003eG\u003c/strong\u003e represent the\u003cem\u003e Nlrp3\u003c/em\u003e expression and serum IL-1β in male mice. Data \u003cstrong\u003eB\u003c/strong\u003e is a Log2-fold change representation of n=3 in each group. Data \u003cstrong\u003eC\u003c/strong\u003e is presented as mean ± SEM of n=4 mice /group and data \u003cstrong\u003eD\u003c/strong\u003e includes n=4 for PBS and n=6 for LPS injected mice, respectively, and analyzed using One-way ANOVA with Kruskal-Wallis test. \u003cstrong\u003eE\u003c/strong\u003e is presented as mean ± SEM of n=3 for each WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e female mice. Data \u003cstrong\u003eF\u003c/strong\u003e (n=3/group) and \u003cstrong\u003eG\u003c/strong\u003e (n=6/group) are presented as mean ± SEM using One-way ANOVA with Bonferroni’s correction.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/addb4e1536bb5c100ccaaadc.jpg"},{"id":108599167,"identity":"a130dd19-d0ed-49d7-89fe-bb9ecb070051","added_by":"auto","created_at":"2026-05-06 11:13:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":333686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice show decreased acute inflammation induced in the KBxN serum transfer arthritis model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, 2 to 2.5-months-old WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were divided in two groups injected with either s C57BL/6 or KBxN serum. \u003cstrong\u003eB\u003c/strong\u003e, On day 0 and day 1, each mouse was i.p. injected with 100 mL of KBxN or C57BL/6 serum. Paw joint inflammation was assessed in \u003cstrong\u003eC,\u003c/strong\u003e male and \u003cstrong\u003eD\u003c/strong\u003e, female mice. Mean arthritis score (MAR) was evaluated in \u003cstrong\u003eE\u003c/strong\u003e, male and \u003cstrong\u003eF\u003c/strong\u003e, female mice, and paw thickness (\u003cstrong\u003eG, H\u003c/strong\u003e) was measured. At the end of the experiment, percentages of Mac2\u003csup\u003eLo \u003c/sup\u003eand Mac2\u003csup\u003eHi \u003c/sup\u003eBMMs was evaluated in all these experimental groups in both \u003cstrong\u003eI\u003c/strong\u003e, male and \u003cstrong\u003eJ\u003c/strong\u003e, female mice. IL-1β levels in the \u003cstrong\u003eK,\u003c/strong\u003e serum and in \u003cstrong\u003eL\u003c/strong\u003e, paw joint lysate is presented for both male and female mice. Data in \u003cstrong\u003eE-J\u003c/strong\u003e are presented as mean ± SEM of n= 5 mice/group for males and 4-5 mice/group for females and were analyzed using Two-way ANOVA. Data \u003cstrong\u003eK\u003c/strong\u003e is presented as mean ± SEM of n=5, 4, 5, 5 (WT, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, WT+KBxN, and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e+KBxN, respectively) for males and n=5, 4, 4, 4 for females. Data in \u003cstrong\u003eL\u003c/strong\u003e is represented as mean ± SEM of n= 5, 4, 5, 5 for males and n= 3/group for female mice. Data in \u003cstrong\u003eK\u003c/strong\u003e and \u003cstrong\u003eL\u003c/strong\u003e were analyzed using One-way ANOVA with Bonferroni’s correction.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/1b9e11e95410b9bd59ef82e7.jpg"},{"id":108599157,"identity":"e7314852-73a8-4423-a5dd-93fe6f463d3b","added_by":"auto","created_at":"2026-05-06 11:13:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":395378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice show mitigated inflammation resulting in reduced severity of bone erosion in response to KBxN serum transfer arthritis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, 2 to 2.5-months-old WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were i.p. injected with 100 mL of serum from KBxN or C57BL/6 mice. Paw joint inflammation, as depicted in \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e for male and female mice respectively was assessed by measuring \u003cstrong\u003eD\u003c/strong\u003e, mean arthritis score and \u003cstrong\u003eE,\u003c/strong\u003e paw thickness at the indicated time points in male mice. \u003cstrong\u003eF \u003c/strong\u003eand\u003cstrong\u003e G\u003c/strong\u003e show mean arthritis score and paw thickness, respectively, for female mice. On day 20, the ankle joints were subjected to μCT, and \u003cstrong\u003eH\u003c/strong\u003e shows the representative 3D images of the ankle joints from WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice from both the sexes treated with C57BL/6 serum or KBxN serum. Data (\u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) are a representation of mean ± SEM of n= 5 for WT+ KBxN and n=6 for \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e+ KBxN mice of both the sexes, analyzed using Two-way ANOVA using Bonferroni’s correction.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/d9907021c8a5dc484275e97b.jpg"},{"id":108599172,"identity":"4e886511-579f-4aea-b4a0-11738cbde6d7","added_by":"auto","created_at":"2026-05-06 11:13:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":331981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emacrophages have high efferocytotic potential and display augmented pro-osteogenic function \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Representative images of Efferocytic macrophages derived from \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003eand littermate control (WT) mice (X200). Apoptotic Ocy454 cells were labeled by CellTracker Deep Dye (red). macrophages were labeled in green. \u003cstrong\u003eB\u003c/strong\u003e, Percentage of efferocytic macrophages derived from both 2- and 7- months old female mice (n= 4 mice/group) were quantified. \u003cstrong\u003eC\u003c/strong\u003e, the percentage of efferocytic macrophages derived from 10-month-old male (n=4 mice/ group) and female (n=4 mice/ group) mice was quantified. BMMs were isolated from 10-weeks old female WT or \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice and co-cultured with 6-months-old WT-BMSCs in osteogenic medium. After 21 days, ALP and Von Kossa staining was performed; representative microscopic images are shown in \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eG,\u003c/strong\u003e quantification is presented in \u003cstrong\u003eE \u003c/strong\u003eand \u003cstrong\u003eF \u003c/strong\u003e(female) and \u003cstrong\u003eH \u003c/strong\u003eand\u003cstrong\u003e I\u003c/strong\u003e (male) (n=3 mice/group for both the sexes) using Image J software. The expression of osteogenic genes, including \u003cem\u003eBglap\u003c/em\u003e,\u003cem\u003e Col1 \u003c/em\u003eand\u003cem\u003e Runx2\u003c/em\u003e was assessed by qPCR in co-cultures from \u003cstrong\u003eJ\u003c/strong\u003e, female and \u003cstrong\u003eK\u003c/strong\u003e, male mice (n=3 mice/group). Data in \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC \u003c/strong\u003eare presented as mean ± SEM and were analyzed using non-parametric t-test and Two-way ANOVA, respectively. Data in \u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eK\u003c/strong\u003e are presented as mean ± SEM and were analyzed using One-way ANOVA with Bonferroni’s correction.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/6cd6fda7b47e700b80642a57.jpg"},{"id":108599154,"identity":"55a9d085-dda6-42de-81a5-313cec343cc7","added_by":"auto","created_at":"2026-05-06 11:13:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":292778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003efemale mice display greater anabolic effects of iPTH on vertebral trabecular bone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4-months old male and female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and WT control littermates were s.c. injected with rh PTH or solvent vehicle for five consecutive days per week, for 4 weeks. \u003cem\u003eIn vivo\u003c/em\u003e mCT assessment of cancellous bone at L5 vertebral bone in (\u003cstrong\u003eA-D\u003c/strong\u003e) female (n=7 WT, n= 8 \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, n=6 WT+ PTH, and n= 5 \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e+ PTH) and (\u003cstrong\u003eE-H\u003c/strong\u003e) male mice (n=6 WT, n= 7 \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, n=5\u0026nbsp; WT+ PTH, and n= 6 \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e+ PTH) were performed. Bone fractional volume, BV/TV; Tb.N, trabecular number; Tb.Th, Trabecular thickness; Tb.Sp, trabecular separation. BMMs were harvested from these mice and the expression of efferocytic macrophage marker genes, including \u003cem\u003eArg1, Il10, Cd36, \u003c/em\u003eand\u003cem\u003e SRA\u003c/em\u003e were assessed by qPCR (n=3 mice/group for both the sexes). Data are presented as Mean ± SEM analyzed using One-way ANOVA post-hoc Tukey’s correction for \u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eH\u003c/strong\u003e and Two-way ANOVA post-hoc Boneferroni’s correction for \u003cstrong\u003eI\u003c/strong\u003e and \u003cstrong\u003eK\u003c/strong\u003e. * p \u0026lt; 0.0332, ** p \u0026lt; 0.0021, *** p \u0026lt; 0.0002, **** p \u0026lt; 0.0001 vs. vehicle-treated WT or \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/1cda153f646b4e24a74f8261.jpg"},{"id":108599153,"identity":"49e266cf-d993-46e1-bad2-6e287ac3e8af","added_by":"auto","created_at":"2026-05-06 11:13:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":337126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic profiling of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGrn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e bone marrow stromal cells (BMSCs) reveal upregulation of cell cycle pathway and downregulation of inhibitory Gi signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand WT -BMSCs (n=3 mice/ group) were allowed to undergo osteogenic differentiation for 21 days and total RNA was subjected to bulk RNAseq analysis followed by REACTOME analysis shows \u003cstrong\u003eA\u003c/strong\u003e, up-regulated and \u003cstrong\u003eB\u003c/strong\u003e, down-regulated pathways in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e-BMSCs as compared to WT-BMSCs.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/8a60031903a4df9e9edcb826.jpg"},{"id":108809916,"identity":"d778a431-b2b5-4663-be62-baf0a7030e25","added_by":"auto","created_at":"2026-05-08 15:56:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2867769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/84cfd46d-8861-43d8-8104-cc694e602608.pdf"},{"id":108599161,"identity":"344b45fc-28f2-4af5-a9b4-7d9f5b2e7979","added_by":"auto","created_at":"2026-05-06 11:13:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":343101,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"SupplementaryFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-9441627/v1/09a994333ccac748bf98b60a.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Loss of Progranulin Expression Decreases NLRP3 Inflammasome-Mediated Inflammation and Enhances Bone Anabolism","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eProgranulin (PGRN), a glycoprotein encoded by the \u003cem\u003eGrn\u003c/em\u003e gene, is constitutively expressed in different tissues, including epithelia, particularly in skin, gastrointestinal tract, reproductive system, immune cells, specific neurons, microglia, and adipose tissue \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Intracellularly, PGRN is in lysosomes, where it regulates protein trafficking, functions as a molecular co-chaperone and helps acidification of lysosomes \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and as secretory protein, PGRN acts as a growth factor \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.It binds to several cell surface receptors such as tumor necrosis factor-α receptor 1 and 2 \u003csup\u003e8\u003c/sup\u003e, Sortilin Related VPS10 Domain Containing Receptor 2 \u003csup\u003e9\u003c/sup\u003e, EGFR \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and Ephrin A2 \u003csup\u003e11\u003c/sup\u003e, to mediate differential cellular functions. Consequently, PGRN plays a pivotal role in embryogenesis, inflammation, wound healing, and tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In humans, a heterozygous loss-of-function (LOF) mutation in \u003cem\u003eGrn\u003c/em\u003e causes Frontotemporal lobar dementia (FTLD/FTD), while homozygous LOF mutation leads to neural ceroid lipofuscinosis type 11 (NCL11), characterized by vision loss, dementia and epilepsy \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePGRN has been widely implicated in immune regulation, particularly in macrophage biology. It inhibits LPS-induced M1 macrophage polarization of RAW264.7 cells, bone marrow-derived macrophages (BMMs), and THP-1 cells \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Consistent with its anti-inflammatory effects, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice undergo spontaneous osteoarthritis and exhibit exacerbated inflamed joints in collagen-induced arthritis (CIA) model \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Moreover, PGRN enhances M2 macrophage polarization of BMMs and RAW264.7 as shown by a decrease in CD86 expression (M1 marker) and increase in CD206 expression (M2 marker) \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, recombinant PGRN increased CD206\u0026thinsp;+\u0026thinsp;macrophages in unilateral ureteral obstruction (UUO) model \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were protected mice from endotoxic shock following LPS injection and showed reduced lung injury \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. All these studies indicated an anti-inflammatory role of PGRN. Conversely, higher serum PGRN levels are reported in RA, OA and Type 2 Diabetes (T2D) individuals. In mice, PGRN mediates high fat diet-induced insulin resistance via IL-6 production in adipose tissue \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Similarly, in severe SLE individuals, higher serum PGRN levels positively correlate with higher serum IL-6 and TNFα \u003csup\u003e19\u003c/sup\u003e. Further, PGRN stimulates secretion of IL-8 by epithelium cells in Multiple Sclerosis (MS), which acts as chemotactic factor for neutrophils and monocytes \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Thus, PGRN is one of those molecules whose imbalance in levels brings out different outcomes depending on the cell type and the diseases being examined, also known as Progranulinopathies, which include autoimmune diseases, metabolic, musculoskeletal and cardiovascular diseases. The dual function of progranulin: as a pro- and an anti- inflammatory molecule, depending on the cellular context is elaborated in the review by Huang et al \u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePreviously, we demonstrated that global deletion of \u003cem\u003eGrn\u003c/em\u003e protects female but not male mice from aging-induced bone loss. A similar phenotype was observed in \u003cem\u003eCx3Cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e female mice, suggesting a critical role of macrophage-derived PGRN in regulating bone homeostasis in female mice \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Macrophages impact bone health, indirectly via inflammation, and directly act as osteoclast precursors, and as osteomacs to enhance or inhibit bone formation functions of osteoblasts depending on the macrophage phenotype \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Additionally, PGRN protects from TNF-ɑ-induced inhibition of osteoblast differentiation and mineralization \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, underscoring its dual role in bone remodeling.\u003c/p\u003e \u003cp\u003eIn FTD individuals with GRN mutations, neuroinflammation due to microglia activation produces inflammatory cytokines including IL-1β and IL-18. These two cytokines are products of NLRP3 inflammasome cascade \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. NLRP3, the NOD-, LRR-, and pyrin domain-containing 3 inflammasome, is a well-known innate immune pathway and is activated by DAMPs and PAMPs. Consequently, caspase-1 is activated which produces bioactive IL-1β and IL-18 to induce inflammatory cell death called pyroptosis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These observations suggest an association of PGRN with NLRP3 inflammasome cascade.\u003c/p\u003e \u003cp\u003eIn the present study we investigated the role of PGRN in influencing macrophage functions which ultimately impact bone health. We find that \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs are less inflammatory than the PGRN-replete BMMs. Mechanistically, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs exhibit down-regulation of \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eIl1β\u003c/em\u003e expression and show poor response to LPS-induced inflammation and M1 polarization. As a result, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice show significant alleviation of inflammation in response to serum transfer-induced arthritis. Moreover, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e female BMMs mice show higher bone anabolic activity in vitro and in response to PTH administration, indicating that PGRN limits the anabolic action of PTH therapy. Mechanistically, our bulk RNA-seq data shows downregulation of G\u003csub\u003ei\u003c/sub\u003e signaling in the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e osteoblasts, which in part, can contribute to enhanced bone formation via activation of Gs signaling in response to PTH. In summary, these results suggest that PGRN expression is associated with NLRP3-inflammasome-mediated inflammation and suppression of bone anabolic functions of macrophages.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eFreshly isolated macrophages from\u003c/b\u003e \u003cb\u003eGrn\u003c/b\u003e \u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eare Mac2\u003c/b\u003e\u003csup\u003e\u003cb\u003ehi\u003c/b\u003e\u003c/sup\u003e \u003cb\u003esubset with decreased inflammatory phenotype\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs reported, female mice with PGRN deficiency in macrophages (\u003cem\u003eCx3Cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice) are protected from aging-induced bone loss \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Thus, we began characterizing the freshly isolated bone marrow macrophages from WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice of both sexes \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We found similar percentages of total macrophages in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice in both sexes expressing CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003e+\u003c/sup\u003e/F4/80\u003csup\u003e+\u003c/sup\u003e/Ly6G\u003csup\u003e+\u003c/sup\u003e/Ly6C\u003csup\u003e+\u003c/sup\u003e. Interestingly, in female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, ~\u0026thinsp;72.35% of these macrophages were Mac2 high (Mac2\u003csup\u003eHi\u003c/sup\u003e, positive for Mac2) and only\u0026thinsp;~\u0026thinsp;23.39% were Mac2 low (Mac2\u003csup\u003eLo\u003c/sup\u003e, negative for Mac2). Conversely, in female WT mice, ~\u0026thinsp;16.18% were Mac2\u003csup\u003eHi\u003c/sup\u003e and ~\u0026thinsp;77.2% were Mac2\u003csup\u003eLo\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Male \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited\u0026thinsp;~\u0026thinsp;94.27% Mac2\u003csup\u003eHi\u003c/sup\u003e and ~\u0026thinsp;3.09% Mac2\u003csup\u003eLo\u003c/sup\u003e macrophages, and male WT showed\u0026thinsp;~\u0026thinsp;25.39% of Mac2\u003csup\u003eHi\u003c/sup\u003e and ~\u0026thinsp;69.4% Mac2\u003csup\u003eLo\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, NanoString-based transcriptomics on FACS-sorted Mac2\u003csup\u003eHi\u003c/sup\u003e subsets displayed enrichment of cell cycle genes likely indicating a higher proliferation rate. Contrarily, Mac2\u003csup\u003eLo\u003c/sup\u003e macrophages showed higher expression of inflammation-related genes, notably, \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eCasp1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. As activation of the NLRP3 inflammasome pathway is sufficient to polarize macrophage towards inflammatory M1 type, we focused on understanding NLRP3 inflammasome signaling in the macrophages. As \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e have higher % of Mac2\u003csup\u003eHi\u003c/sup\u003e subset, we assessed NLRP3 inflammasome pathway in M-CSF dependent bone marrow-derived macrophages (BMMs) from WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. We found that female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT BMMs displayed reduced expression of \u003cem\u003eNlrp3\u003c/em\u003e, \u003cem\u003eIl1\u0026szlig;\u003c/em\u003e, \u003cem\u003eTnfa\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. However, male BMMs showed decreased \u003cem\u003eIl1\u0026szlig;\u003c/em\u003e with other genes remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Additionally, female WT BMMs treated with rm PGRN for 24 hrs greatly enhanced the expression of \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eIl1\u0026szlig;\u003c/em\u003e genes but not in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. These findings suggest an upstream regulatory role of PGRN in the expression of \u003cem\u003eNlrp3\u003c/em\u003e pathway genes in female mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrn\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice exhibit a suppressed inflammatory response to LPS administration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e results suggested that macrophages from \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice have lower \u003cem\u003eNlrp3\u003c/em\u003e expression and possibly reduced NLRP3 inflammasome signaling. To corroborate our \u003cem\u003ein vitro\u003c/em\u003e findings, we challenged female and male WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with LPS (15 mg/kg body wt.) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). BMMs were prepared and subjected to NanoString-based transcriptome profiling. We observed that female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs showed differentially expressed genes with more than \u0026gt;\u0026thinsp;50-fold increase in the expression of \u003cem\u003eCcl2\u003c/em\u003e and \u003cem\u003eCcl7\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which are macrophage chemotactic proteins that also play a role in macrophage polarization. Of interest, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT BMM showed reduced expression of \u003cem\u003eNlrp3\u003c/em\u003e in response to LPS, which was further validated by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Given that IL-1β production is largely NLRP3 inflammasome dependent, we indeed found that the serum IL-1β was lower in the LPS challanged \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs.WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Reportedly, NLRP3 inflammasome signaling is involved in M1-type macrophage polarization. We found that LPS challenged female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT BMMs displayed down-regulation of M1-type genes including \u003cem\u003eiNOS, Tnfa, Fpr2, Cxcl10\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Similarly, a reduced \u003cem\u003eNlrp3\u003c/em\u003e expression and less serum IL-1β levels in the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT was observed male mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cb\u003eG\u003c/b\u003e). Taken together, these results suggest that PGRN expression positively correlates with activation of the LPS-induced NLRP3 inflammasome signaling cascade, which in turn is required for M1-type macrophage polarization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrn\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice show delayed onset of inflammation in response to STA-induced arthritis.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe investigated the functional relevance of the association of PGRN with NLRP3-inflammasome dependent IL-1β production in the serum-transfer (STA)- induced rheumatoid arthritis (RA) mouse model \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Accordingly, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice with KBxN or control serum, and arthritis score and paw thickness was measured from until day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Both male and female WT mice injected with KBxN serum showed robust inflammation at each time point until day 5 as observed by an increase in mean arthritis score (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F\u003cb\u003e)\u003c/b\u003e and paw thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Strikingly, both \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed significantly lower arthritis scores and paw thickness compared to sex-matched WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-H).\u003c/p\u003e \u003cp\u003eTo understand the cellular mechanism, freshly isolated bone marrow macrophages were immune-phenotyped on day 5. It was observed that the relative percentages of Mac2\u003csup\u003eHi\u003c/sup\u003e and Mac2\u003csup\u003eLo\u003c/sup\u003e subsets in both the sexes of WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e genotypes remained unchanged, irrespective of whether the mice received control or KBxN serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI \u003cb\u003eand J\u003c/b\u003e). Furthermore, we found that the levels of IL-1β were significantly higher in serum and ankle joint lysates from the WT vs. \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e arthritic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK \u003cb\u003eand L\u003c/b\u003e) and in both sexes. Our data provided important information that in the absence of PGRN both \u003cem\u003eNlrp3\u003c/em\u003e gene expression and IL-1β cytokine levels are downregulated consistent with the attenuation of inflammation in STA model of RA in the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Thus, PGRN likely promotes inflammation via promoting the NLRP3 inflammasome cascade in WT mice subjected to STA induced RA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrn\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emice show mitigated inflammation during later stages of STA-induced arthritis and attenuated bone erosion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn STA model of RA, inflammation starts to resolve gradually after day 5\u0026ndash;7 and bone erosion is detectable by day 20 \u003csup\u003e26, 28\u003c/sup\u003e. Therefore, we evaluated the dynamics of inflammation at later stages of RA and bone phenotype as the RA progresses in both the WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice of both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT mice showed mitigation of inflammation throughout the progression of RA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u003cb\u003eand C)\u003c/b\u003e, evident by a significantly reduction in mean arthritic score and paw thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G). Furthermore, on day 20, WT arthritic mice showed bone erosion around articular surfaces at the ankle joints, as assessed by \u0026micro;CT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Strikingly, arthritic \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed visible protection of erosive bone loss as compared to WT arthritic counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u003cb\u003e).\u003c/b\u003e In female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, we did see a similar pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Taken together, these data suggest that PGRN deficiency in mice reduces the severity of erosive bone loss in STA-induced RA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrn\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eBMMs are highly efferocytic and enhance osteogenic differentiation of bone marrow stromal cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the current study, we found that \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs are mostly galectin3\u003csup\u003e+\u003c/sup\u003e (Mac2\u003csup\u003eHi\u003c/sup\u003e). Since galectin3 is reported to enhance the efferocytic potential of human monocytes in phagocytosing apoptotic neutrophils \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we assessed the efferocytic potential of PGRN-deficient BMMs. This was achieved by co-culturing labelled apoptotic OCY545 cells with BMMs from either WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice for 24 hrs and later counting the labelled BMMs (a read out of efferocytosis) using fluorescence microscopy. We found that \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs showed a higher percentage of labeled cells (red) due to increased efferocytosis of apoptotic OCY545 as compared to WT BMMs at 2 months and 7 months of age, and in both the sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). These results suggest that PGRN limits macrophage efferocytic potential, possibly via downregulation of Mac2 expression.\u003c/p\u003e \u003cp\u003eIt is well established that osteal macrophages are critical to bone formation via different mechanisms, including depletion of apoptotic osteoblasts and by the recruitment of osteoblast precursors \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Also, the osteogenic differentiation of bone marrow stem cells is influenced by osteal macrophages and further influenced by macrophage phenotype, with M1 being inhibitory and M2 with stimulatory actions \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We assessed the potential of PGRN deficient BMMs to regulate bone marrow stromal cell osteogenic differentiation. Female WT BMSC co-cultured with WT BMMs displayed enhanced ALP\u003csup\u003e+\u003c/sup\u003e area and Von Kossa\u003csup\u003e+\u003c/sup\u003e area as compared to the control WT BMSC alone cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. However, intriguingly, female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs co-cultured with WT-BMMs exhibited an increased ALP\u003csup\u003e+\u003c/sup\u003e and Von Kossa\u003csup\u003e+\u003c/sup\u003e areas compared with either the WT BMM\u0026thinsp;+\u0026thinsp;WT BMSC co-culture or WT BMSC alone cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E \u003cb\u003eand F)\u003c/b\u003e. This suggests that BMMs from female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice have greater pro-osteogenic activity than BMMs from female WT mice. In the co-culture experiments using male mice, WT BMMs did enhance the osteogenic differentiation of BMSCs as compared to BMSC cultures with no BMMs added to them, as assessed by the increase in the percent ALP\u003csup\u003e+\u003c/sup\u003e and Von Kossa\u003csup\u003e+\u003c/sup\u003e areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, H \u003cb\u003eand I)\u003c/b\u003e. However, BMMs from male \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not display enhanced pro-osteogenic activity compared to BMMs from male WT mice. We further confirmed these observations using qPCR-based relative gene expression of osteogenic marker genes, including \u003cem\u003eAlp, Runx2, Col1, and Ocn.\u003c/em\u003e Gene expression studies of the co-cultures clearly showed that female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMM have higher pro-osteogenic activity than the WT-BMMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), while male \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs did not further enhance the expression of osteogenic genes as compared to BMM from WT male counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Taken together, these results suggest that the PGRN deficient BMMs derived from female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice have greater pro-osteogenic supporting potential than WT BMMs or male \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFemale\u003c/b\u003e \u003cb\u003eGrn\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice display greater bone anabolic action of iPTH\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn our current study, we observed that BMMs derived from female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed greater pro-osteogenic function in driving the osteogenic differentiation of BMSCs. Also, it has been reported that macrophages are essential for bone anabolic actions of iPTH therapy in mice \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, we evaluated whether PGRN-deficient macrophages are different in responses to iPTH therapy-induced bone anabolic actions \u003cem\u003ein vivo\u003c/em\u003e and if sex-differences are observed. well. To achieve this, we s.c. injected both WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with PTH (80 \u0026micro;g/kg/day) for 5 days/ week for 4 weeks. The L\u003csub\u003e5\u003c/sub\u003e vertebrae were subjected to bone phenotyping using mCT after 4 weeks of PTH treatment. We observed that administration of PTH induced bone formation in both male and female WT mice and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice as shown by a significant increase in BV/TV, Tb.Th., and Tb.N. and a decrease in Tb.Sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-H). Notably, PTH-injected \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e female mice (\u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e+PTH) had an increased anabolic response to iPTH compared to their female WT counterparts injected with PTH (WT\u0026thinsp;+\u0026thinsp;PTH), as shown by a higher BV/TV, Tb.Th., and Tb.N. with a significant decrease in Tb.Sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). However, PTH-injected \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male mice (\u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e+PTH) showed similar parameters of trabecular bone to male WT counterparts injected with PTH (WT\u0026thinsp;+\u0026thinsp;PTH). These data are consistent with our \u003cem\u003ein vitro\u003c/em\u003e findings of the pro-osteogenic function of PGRN-deficient macrophages. Furthermore, immunostaining of F4/80\u003csup\u003e+\u003c/sup\u003e osteal macrophages on the femur bone surface of the WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not show any difference in osteal macrophage number in any of the groups or between the sexes (\u003cb\u003eSuppl. Figure\u0026nbsp;1\u003c/b\u003e). Moreover, BMMs derived PTH injected mice, both female and male exhibited a pattern of increased expression of efferocytic macrophage marker genes, including \u003cem\u003eArg1\u003c/em\u003e, \u003cem\u003eIl10\u003c/em\u003e, \u003cem\u003eCd36\u003c/em\u003e and \u003cem\u003eSRA\u003c/em\u003e, irrespective of the sex and genotype.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptomic profiling of female\u003c/b\u003e \u003cb\u003eGrn\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ebone marrow stromal cells (BMSCs) reveal downregulation of inhibitory Gi signaling pathway\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present study, we found that female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs have greater potential of promoting osteogenic differentiation of BMSCs \u003cem\u003ein vitro\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e PTH stimulated higher bone anabolic effects in female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Thus, we performed RNAseq on the BMSCs after 21 days of osteogenic differentiation from both WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e to understand any intrinsic differences. We found that \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMSCs showed\u0026thinsp;\u0026gt;\u0026thinsp;5-fold increase in expression of genes of cell cycle progression, and \u0026gt;\u0026thinsp;3-fold decrease in the expression of genes that belong to inhibitory Gi signaling. Further Reactome analysis indeed showed upregulation of cell cycle genes and down-regulation of genes involved in Gi signaling in the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMSCs vs. WT-BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). We know from our previous work that inhibition of Gi signaling with pertussis toxin, using genetic mouse models, enhances bone formation in aging females, and accelerates bone anabolic action of PTH only in female mice \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The resulting active Gsα signaling pathway, which is a known PTH target, mediates the bone formation function of osteoblasts.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAvailable reports indicate PGRN has dual action, acting as a pro- or anti-inflammatory molecule depending on the cellular context \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Full-length PGRN can be digested by proteases, both intracellularly or extracellularly, into different granulins including, G, F, B, A, C, D, and E, which are known to play pro-inflammatory roles \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Reportedly, PGRN is anti-inflammatory in mouse models of osteoarthritis (OA) and collagen-induced arthritis (CIA) and is attributed to its potential to block the TNF-ɑ pathways by blocking its receptors (TNFRs) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Similarly, Attstrin, PGRN analog, is anti-inflammatory \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In addition to its anti-inflammatory role, PGRN is essential for efficient osteoclast differentiation as the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e OC precursors are resistant to RANKL- induced osteoclast formation \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Additionally, PGRN protects from the inhibitory action of TNF-α on osteoblasts differentiation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Previously, we reported that female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e are resistant to aging-associated bone loss. This is partly attributed to higher bone formation rate and reduced osteoclastic bone resorption. However, male \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice do not exhibit such an aging phenotype and continue to lose bone despite reduced osteoclastic bone resorption. However, the mechanism(s) underlying the female-sex specific aging-associated bone protective role of PGRN is not clear. The female-specific bone protective role of PGRN was also seen in \u003cem\u003eCx3Cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, suggesting that the negative role of PGRN on bone mass results from production of the protein by macrophage lineage cells. Thus, in the present study we investigated the role of PGRN in mediating the effects of macrophages on bone homeostasis.\u003c/p\u003e \u003cp\u003eFirstly, to understand the role of PGRN in macrophage biology, we characterized freshly isolated bone marrow macrophages \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We found that \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice have a higher percentage of Mac2\u003csup\u003eHi\u003c/sup\u003e macrophages in both male and female mice, suggesting that PGRN may have a role in the regulation of Mac2 expression. Mac2, also known as galectin3, is upregulated in microglia of patients with haploinsufficiency due to loss-of-function mutation in \u003cem\u003eGrn\u003c/em\u003e genes. Galectin3 has been reported to be essential for human macrophage invasion and for suppressing pro-inflammatory cytokine production \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Interestingly, our transcriptomics data indeed revealed that Mac2\u003csup\u003eHi\u003c/sup\u003e macrophages express low levels of inflammation-related genes, and notably, we found that \u003cem\u003eNlrp3\u003c/em\u003e was downregulated in these PGRN-deficient macrophages. As NLRP3 inflammasome signaling is involved in the maturation and secretion of IL-1β, we found that the serum IL-1β levels and \u003cem\u003eNlrp3\u003c/em\u003e expression in the M-CSF dependent BMMs were lower in the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice as compared to the WT in response to LPS challenge, thus suggesting that PGRN expression is positively correlated with NLRP3 inflammasome signaling pathway gene expression.\u003c/p\u003e \u003cp\u003eTo understand the disease relevance of these findings, we employed a serum transfer-induced rheumatoid arthritis (RA) model. This model of RA is principally dependent on IL-1β-mediated inflammation and bone loss. The results clearly indicated that PGRN presence is required for effective NLRP3 mediated-IL-1β-induced inflammation as the \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e showed signs of alleviated inflammation in the paw in both the sexes and that the mice showed reduced severity of bone erosion. The reduced serum and paw joints levels of IL-1β in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e further suggests that PGRN regulates NLRP3 inflammasome signaling leading to IL-1β-mediated inflammation, arthritis and the subsequent bone erosion in WT mice.\u003c/p\u003e \u003cp\u003eIn the present study, M-CSF dependent BMMs from both male and female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated enhanced efferocytotic activity \u003cem\u003ein vitro\u003c/em\u003e. Increased efferocytotic activity of macrophages has been reported to be associated with increased bone formation \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, which is due to clearance of apoptotic mature osteoblasts and recruiting new osteoblast precursors at the site of bone formation. These efferocytic macrophages are reported to be F4/80\u003csup\u003e+\u003c/sup\u003e osteomacs present on the bone surface. Further, Cho et al. in 2014 reported that these F4/80\u003csup\u003e+\u003c/sup\u003e osteomacs are critical for the anabolic action of intermittent parathyroid hormone therapy (iPTH) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Interestingly, we found that BMMs from female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e co-cultured with bone marrow stromal cells (BMSCs) from WT mice showed greater osteogenic differentiation potential than the WT BMMs or BMMs alone However, BMMs from male \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e co-cultured with bone marrow stromal cells (BMSCs) did not show enhanced osteogenic differentiation from WT counterparts. Further, \u003cem\u003ein vivo\u003c/em\u003e, we found that the female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed enhanced anabolic action of iPTH in L5 vertebrae as compared to their WT counterparts. These results further highlight the importance of macrophage efferocytotic function as an essential process of iPTH therapy. However, for reasons unknown, in our study the enhanced anabolic effect of iPTH was limited only to female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e despite increased efferocytic function in both the sexes \u003cem\u003ein vitro\u003c/em\u003e. Further, the transcriptomics and reactome analysis of BMSCs from \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e revealed downregulation of genes involved in the Gi signaling pathway. Intriguingly, Gi signaling has been well documented to hamper bone formation and to limit the anabolic action of iPTH therapy in female mice \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. One such Gi-GPCR is \u003cem\u003eHtr1b\u003c/em\u003e which was down-regulated in the female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMSCs. It is well-documented that gut-derived serotonin binds to \u003cem\u003eHtr1b\u003c/em\u003e on osteoblasts and inhibits bone formation via decreasing cAMP response element-binding protein (CREB) function, a key transcription factor that promotes osteoblast proliferation and differentiation \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, an increased pro-osteogenic and efferocytic functions of macrophages together with inhibition of Gi signaling in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e females can partly explain the enhanced bone anabolic action of iPTH therapy. This could possibly explain our previous finding that only female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice are resistant to aging-associated bone loss \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, further mechanistic studies are required to understand the sexual dimorphic actions of PGRN on aging-induced bone loss.\u003c/p\u003e \u003cp\u003eIn summary, deficiency of PGRN expression in mouse macrophages downregulates \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eIl1β\u003c/em\u003e expression to alter NLRP3 signaling cascade which confers macrophage with less inflammatory phenotype. Consequently, the severity of bone erosion is reduced in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e serum transfer-induced RA model in mice. Furthermore, these PGRN-deficient macrophages exhibit enhanced osteogenic potential that can contribute to greater anabolic action of PTH in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e female mice and protect these mice from aging-associated bone loss. However, molecular mechanisms governing progranulin-mediated activation of the NLRP3 inflammasome and its bone anti-anabolic effects are unclear and will be of great interest for further studies.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal studies were approved by and performed in accordance with the Institutional Animal Care and Use Committees at the San Francisco VA Medical Center and the University of California, San Francisco (UCSF). We bred heterozygous \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/+\u003c/em\u003e\u003c/sup\u003e mice in C57BL/6 background, generously provided by Dr. Robert V. Farese at UCSF, to generate \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT littermate (used as controls). KRN mice, provided by Dr. Clifford Lowell at UCSF, were bred with NOD mice (The Jackson Laboratory, Bar Harbor, ME) to generate K/BxN mice. \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice of both sexes and various ages were used in different experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of mouse bone marrow-derived macrophage and culture of M-CSF dependent macrophages (BMMs)\u003c/h3\u003e\n\u003cp\u003eFreshly isolated bone marrow macrophages were immune-phenotyped (flow cytometry section) or cultured with M-CSF to generate M-CSF-dependent macrophages (BMMs) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were euthanized and hindlimb bones were excised, demuscled, and the epiphyseal ends were cut open, and the marrow was flushed with RPMI-1640 (Gibco) using a syringe with a needle size of 26\u003csup\u003e1/2\u003c/sup\u003e gauge. Upon RBCs lysis using Lysis Buffer (Cat#00-4333-57, eBioscience), cells were washed and resuspended in RPMI-1640 growth medium containing 10% FBS, 1% penicillin-Streptomycin, and 0.1% Fungizone, and 20 ng/mL macrophage-colony stimulating factor (M-CSF; Cat# 416-ML-050/CF, R\u0026amp;D Systems) for 24 hours. Next day, the non-adherent fraction is collected and cultured in RPMI-1640 growth medium with M-CSF (20 ng/mL) for 6 days to generate macrophages (BMMs).\u003c/p\u003e \u003cp\u003eTo understand the effect of PGRN deficiency or action of exogenous PGRN on the expression of the proinflammatory genes, BMMs were prepared from 12-weeks-old \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and their littermate WT. The BMMs were treated for 24 hrs with 500 ng/mL of recombinant mouse PGRN (Cat# AG-40A-0189Y-C010, AdipoGene Life Sciences).\u003c/p\u003e\n\u003ch3\u003eBone marrow stromal cells (BMSCs) isolation and co-culture with BMMs\u003c/h3\u003e\n\u003cp\u003eBone marrow macrophages (BMMs) were prepared from 10-month-old \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e or WT mice as described above. On the day before the co-culture assay, male and female C57BL/6 mice of 10 months age were used for isolating bone marrow stromal cells (BMSCs). In brief, total bone marrow was flushed out and BMSCs were enriched with MACS technology by depleting the mature hematopoietic lineage cells with CD11b\u003csup\u003e+\u003c/sup\u003e MACS microbeads (Cat#130-126-725, Miltenyi Biotec). The enriched BMSCs were plated in a 6-well plate at a density of 3 x 10\u003csup\u003e6\u003c/sup\u003e cells/well for female cell BMSCs and 2.6 x 10\u003csup\u003e6\u003c/sup\u003e cells /well for male BMSCs (day 0). On day 1, bone marrow macrophages (BMMs) were enzymatically freed and then seeded into the culture wells containing BMSCs in a ratio of 1:7 (BMMs/BMSCs). The cultures were maintained undisturbed for 5 days in a 5% CO\u003csub\u003e2\u003c/sub\u003e maintained at 37\u0026deg;C. culture medium was removed along with all non-adherent cells and replaced with fresh alpha MEM with 50 \u0026micro;g/ml ascorbic acid and 3 mM β-glycerophosphate to initiate osteogenic differentiation. The culture medium was replaced every three days. At day 21, alkaline phosphatase and Von Kossa stainings were performed using ALP staining kit (Cat# ab284936, Abcam) and Silver Nitrate staining methods, respectively. Stained areas were quantified using NIH Image J software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, cDNA preparation and qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the cells using TRI reagent-based phenol-chloroform isolation followed by purification with RNeasy mini kit (Cat# 74104, Qiagen), according to the manufacturer\u0026rsquo;s instructions. The isolated Total RNA was used to prepare cDNA using TaqMan\u0026trade; Reverse Transcription Reagents (Cat# N8080234, Thermo-Fisher) according to the manufacturer's instructions. qPCR was performed using 10 ng of cDNA using SYBR\u0026trade; Green Universal Master Mix and 100 nM of forward and reverse primer pairs for each gene, designed using Primer Bank. \u003cem\u003eGapdh\u003c/em\u003e was used as an endogenous control gene, and the relative expression of the gene was calculated using 2\u003csup\u003e\u0026minus;\u0026thinsp;dCT\u003c/sup\u003e. Primers used for all genes are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of all qPCR primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward Primer (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse Primer (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNlrp3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGA AGA GAC CAC GGC AGA AG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCT TGG ACC AGG TTC AGT GT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIl1β\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTG CAA GTG TCT GAA GCA GC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAA AGG TTT GGA AGC AGC CC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTnfα\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCC TCC CTC TCA TCA GTT CTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGC AGC CTT GTC CCT TG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIl6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATC CAG TTG CCT TCT TGG GAC TGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAA GCC TCC GAC TTG TGA AGT GGT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eiNOS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAG ACA GGG AAG TCT GAA GCA C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCA GCA GTA GTT GCT CCT CTT C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFpr2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAG CCT GGC TAG GAA GGT G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGC TGA AAC CAA TAA GGA ACC TG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCxcl10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCA AGT GCT GCC GTC ATT TTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGC TCG CAG GGA TGA TTT CAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBglap\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTG ACC TCA CAG ATG CCA AG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTA GCG CCG GAG TCT GTT C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCol1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCG AAG GCA ACA GTC GCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTT GGT GGT TTT GTA TTC GAT GAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRunx2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGA GAC CAA CCG AGT CAT TT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACG CCA TAG TCC CTC CTT TT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eArg-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGT CCC TAA TGA CAG CTC CTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCA TCC ACC CAA ATG ACA CAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIl10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTG GAC AAC ATA CTG CTA ACC G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGG CAT CAC TTC TAC CAG GTA A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCd36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATT AAT GGC ACA GAC GCA GC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCA TTG GCT GGA AGA ACA AA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSRA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTC GGG ATC TCC TGG ACC TA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATC CCA GCG ATC ATC ACA GA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGapdh\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGC ACC ACC AAC TGC TTA G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGA TGC AGG GAT GAT GTT C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eFreshly isolated adult mouse bone marrow macrophages were characterized as described previously \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In brief, freshly isolated bone marrow cells after RBS lysis were incubated with TruStain FcX to block the Fc receptors and cells were stained with fluorescence tagged antibodies as listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. BD compensation beads were used for setting up compensation. Samples were acquired using BD FACSAria\u0026trade; FUSION and populations were sequentially gated via FMOs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of antibodies used for flow cytometry\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatalog\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTruStain FcX (Clone 93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e101320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePacific Blue anti-mouse CD45 (clone 30-F11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e103126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAllophycocyanin (APC) F4/80 (clone BM8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e123116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPC-Cy7 rat anti-CD11b (clone M1/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e557657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePE/Dazzle 594 Ly-6G (clone1A8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e127648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpark UV\u0026trade; 387 anti-mouse Ly-6C Antibody (clone HK1.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e128059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa Fluor 488 Mac2 (clone M3/38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e125410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eNanoString nCounter\u003c/h3\u003e\n\u003cp\u003e7-months-old male and female C57BL/6 mice were used to evaluate the differential transcriptome of Mac2\u003csup\u003eLo\u003c/sup\u003e vs Mac2\u003csup\u003eHi\u003c/sup\u003e in freshly isolated macrophages population. In brief, freshly isolated mouse macrophages were enriched with CD11b\u003csup\u003e+\u003c/sup\u003e MACS microbeads, incubated with Fc block, and then stained for Mac2 antibody (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) to sort Mac2\u003csup\u003e+\u003c/sup\u003e cells with high (Mac2\u003csup\u003eHi\u003c/sup\u003e) and low Mac2 (Mac2\u003csup\u003eLo\u003c/sup\u003e) with a BD FACSAria\u0026trade; FUSION at the SF VAMC FACS core. RNA was extracted from the sorted Mac2\u003csup\u003eLo\u003c/sup\u003e and Mac2\u003csup\u003eHi\u003c/sup\u003e cells with an Invitrogen PureLink RNA Micro Scale kit (Waltham, MA). Similarly, RNA from M-CSF dependent BMMs harvested from LPS-challenged WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e were subjected to NanoString nCounter system (Seattle, WA) using a nCounter\u0026reg; Myeloid Innate Immunity Panel.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLPS challenge experiment\u003c/h2\u003e \u003cp\u003eMale and female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT littermates of 6-9-months-age were i.p. injected with 15 mg/kg Lipopolysaccharide (LPS) (Cat# L4130, Escherichia coli O111:B4, Sigma, MO). After 6 hrs, the mice are euthanized, bone marrow was collected and M-CSF dependent BMMs were prepared. Subsequently, BMMs were subjected to NanoString-based gene expression analysis. Blood serum was also collected for cytokine level measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInduction of serum-transfer-induced (STA) rheumatoid arthritis\u003c/h2\u003e \u003cp\u003eWe bred the KRN mice with NOD mice to obtain the K/BxN mice. The blood is collected from K/BxN mice at the age of 8\u0026ndash;10 weeks old and allowed to clot at RT for 15 min and then centrifuged at 2000 x g for 10 min at 4\u0026deg;C. The KBxN serum is then stored in -80 until use. We used the serum from C57BL/6 mice of the same age as control serum.\u003c/p\u003e \u003cp\u003eThe experiment involved injection of 100 \u0026micro;L of K/BxN serum or control serum in 2- months-old WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male and female mice. Two injections were done one on day 0 and on day 1 i.p. As a measure of inflammation, arthritis score and paw thickness were measured on each day until day 5 post first injection for the inflammatory phase study. For the effector phase study, the inflammation was monitored every three days until day 15 \u003csup\u003e26, 28\u003c/sup\u003e. After euthanizing the mice, serum was collected, one ankle joint from each mouse was snap frozen for ELISA. The femurs were used to characterize the BMMs using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of arthritis score and paw thickness\u003c/h2\u003e \u003cp\u003eTo measure inflammation in the RA mice, we used the arthritis scoring method as previously described \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. For each of the four limbs (maximum four points per limb, up to a combined total of 16), score points (1\u0026ndash;4) according to the presence of the feature with the greatest point value.1 point if there is only redness of the bottom of the footpad; 2 points if there is visible thickening of the paw, 3 points if the swelling of the ankle is sufficient to make the ankle equal to or greater in width than the mid footpad, 4 points if there is swelling of at least one digit. The paw thickness was measured using digital calipers at the ankle joint (malleoli) and is presented in millimeters (mm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIntermittent Parathyroid hormone (iPTH) administration\u003c/h2\u003e \u003cp\u003e4-months-old WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice of both the sexes were injected s.c. with recombinant human PTH 1\u0026ndash;34 (Bachem Inc., CA) (dissolved in 10 mM acetic acid in PBS with 2% heat inactivated C57BL/6 serum) at a dose of 80 \u0026micro;g/kg body weight for five consecutive days per week, for 4 weeks. Control animals were treated with the same volume of vehicle \u0026ndash; 10% acetic acid PBS in PBS containing 2% heat-inactivated C57BL/6 serum. At the end of the experiment, mice were euthanized and L5 vertebrae and hind limbs were fixed in 4% PFA and then subjected to micro-CT (\u0026micro;CT) analysis for bone phenotyping.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicro-computed Tomography\u003c/h2\u003e \u003cp\u003eHind limb paws with intact ankle joint from the RA mice and L5 vertebra from PTH-treated mice were fixed in 4% PFA for 48 hours at 4\u0026deg;C and then stored in 70% ethanol before being assessed using \u0026micro;CT scan and histomorphometry. Ankle joints were scanned using a Scanco VivaCT-50 \u0026micro;CT system (Scanco Medical, Br\u0026uuml;ttisellen, Switzerland) with an X-ray energy of 55 kV, a voxel size of 10.0 \u0026micro;m, and an integration time of 500 ms. We analyzed the 3D images of the whole ankle joint and visually assessed the bone erosion. For the PTH study, the trabecular region of interest (ROI) within L\u003csub\u003e5\u003c/sub\u003e was assessed. The ROI was defined as a cylindrical volume of 0.5 mm\u0026sup2; cross-sectional area and trabecular parameters were evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSerum and paw joint IL-1β measurement\u003c/h2\u003e \u003cp\u003e To assess the levels of cytokines in the serum in LPS-injected animals, we used a flow cytometry-bead based immunoassay using LegendPlex\u0026trade; Mouse M1 Macrophage Panel (8-plex) (Cat#740848, Biolegend), according to the kit instructions. The concentration of each cytokine is subsequently determined by referencing a standard curve generated concurrently within the same assay using online LEGENDplex\u0026trade; Data Analysis Software by applying a 5-parameter curve fitting algorithm. For serum-transfer induced arthritis studies, serum and paw-joint lysate IL-1β was measured using Mouse IL-1 beta/IL-1F2 DuoSet ELISA kit (Cat# DY401-05) following manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eTibiae were collected from PTH or vehicle treated WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and fixed in 4% PFA followed by demineralization. 5 \u0026micro;m thick sections were made using cryotome and subjected to IHC. Briefly, the sections were blocked with 5% donkey serum in PBS and then incubated with primary antibody against F4/80 (Cat# MF48000) overnight at 4\u003csup\u003eο\u003c/sup\u003eC in a humidified chamber. Next day, the sections are washed and incubated with Goat anti-rat Alex Flour\u0026trade; 555 (Cat# A-21434). The slides were analyzed at 20X magnifications using the microscope BZ-X800 (Keyence).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNAseq\u003c/h2\u003e \u003cp\u003eBMSCs from WT and \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e female mice (n\u0026thinsp;=\u0026thinsp;3/genyotype) underwent osteogenic differentiation as described before. At day 21, RNA was isolated and RNAseq analysis was performed by Novogene using NovaSeq PE150 platform. Pathway analysis was carried out using the Reactome database and differentially regulated genes were assessed using threshold is normally set as: p adj\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out using Graph pad Prism 10 software (version 10.6.1). For comparison of two groups, we used an unpaired student\u0026rsquo;s t-test. For experiments with more than 2 groups, we used one-way ANOVA with post-hoc Bonferroni\u0026rsquo;s correction and for more than 2 parameters, we used Two-way ANOVA. Each graph presented is a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTERESTS\u003c/h2\u003e \u003cp\u003eAll authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eVP, LW, MN, GM and RAN, designed and planned the study. VP, LW, GK, PN, YW and CW performed all the experiments. VP and LW analyzed all data and performed statistical analysis. VP wrote the first draft which was later corrected by all the co-authors and approved the final version.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThe authors would like to thank San Francisco VA Medical Center (SF VAMC) Bone Core for its technical assistance. This work was supported by the Veterans Affairs Merit Review Program (5I0BX003213 to R.A.N.), NIH P30 (2P30AR075055). The work was previously presented in parts as a poster at the American Society for Bone and Mineral Research Annual Meeting 2024 (Toronto, CA) and 2025 (Seattle, USA).\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eAll datasets generated during and/or analyzed during the current study are presented in the article. Any additional raw files of transcriptomics are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBateman A, Bennett HP. The granulin gene family: from cancer to dementia. Bioessays. 2009;31(11):1245\u0026ndash;54. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/bies.200900086\u003c/span\u003e\u003cspan address=\"10.1002/bies.200900086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Review.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmid A et al. Role of progranulin in adipose tissue innate immunity. 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Cell. 2008;135(5):825\u0026ndash;837. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell\u003c/span\u003e\u003cspan address=\"10.1016/j.cell\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 2008.09.059\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Progranulin, macrophages, inflammation, NLRP3, inflammasomes, IL-1β, arthritis, iPTH","lastPublishedDoi":"10.21203/rs.3.rs-9441627/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9441627/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProgranulin (PGRN), a glycosylated protein, is expressed in most tissues, including bones, and its level is elevated in the serum and joints of individuals with inflammatory bone loss disorders such as rheumatoid arthritis (RA). Previously, using global and macrophage-specific \u003cem\u003eGrn\u003c/em\u003e deletion mice, we demonstrated that loss of PGRN protects against age-related bone loss selectively in females, suggesting a sex-dependent role for macrophage-derived PGRN in skeletal homeostasis. Here, we investigated the role of PGRN in the regulation of macrophage-mediated inflammation and bone formation. Immune-phenotyping revealed that \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT mice exhibited higher percentages of Mac2\u003csup\u003eHi\u003c/sup\u003e subsets in both sexes. Transcriptomic analysis of Mac2\u003csup\u003eHi\u003c/sup\u003e vs. Mac2\u003csup\u003eLo\u003c/sup\u003e from WT mice showed reduced expressed expression of \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eIl1β\u003c/em\u003e at baseline in both sexes. Further, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs. WT M-CSF-dependent macrophages (BMMs) revealed decreased expression of \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eIl1β\u003c/em\u003e following LPS challenge in vivo in both sexes. Consistent with this, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed markedly reduced IL-1β production in serum and paw joints, and attenuated bone erosion in the STA-induced RA model, indicating altered NLRP3 inflammasome signaling in \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Notably, PGRN deficiency enhances the osteoanabolic capacity of macrophages: female \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BMMs potentiated osteogenic differentiation of mesenchymal stem cells, and \u003cem\u003ein vivo\u003c/em\u003e, \u003cem\u003eGrn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e females exhibited higher trabecular bone formation in response to intermittent PTH. Collectively, PGRN deficiency in BMMs is negatively associated with \u003cem\u003eNlrp3\u003c/em\u003e expression and IL-1β production and causes reduced inflammation and bone erosion in mice subjected to STA-induced RA. Furthermore, PGRN limits the bone-anabolic action of PTH in a female sex-specific manner.\u003c/p\u003e","manuscriptTitle":"Loss of Progranulin Expression Decreases NLRP3 Inflammasome-Mediated Inflammation and Enhances Bone Anabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 11:12:31","doi":"10.21203/rs.3.rs-9441627/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-06T13:08:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-02T11:27:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-28T02:06:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T02:30:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T19:26:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bone Research","date":"2026-04-16T19:26:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9fb21136-f1a4-4a9c-a18e-0ddf661e4954","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-06T13:08:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-02T11:27:57+00:00","index":1,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67125772,"name":"Biological sciences/Physiology/Bone"},{"id":67125773,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-05-06T11:12:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 11:12:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9441627","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9441627","identity":"rs-9441627","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}