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Yuzhao Liu,1,* Jingru Cai,2,* Yangang Wang,1 Xiangli Zhao,3 Yun Qiao,4 Chuan-Ju Liu3 1Department of Endocrinology, the Affiliated Hospital of Qingdao University, Qingdao, People’s Republic of China; 2Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 3Department of Orthopaedic Surgery, New York University Grossman School of Medicine, New York, NY, USA; 4Department of Traditional Chinese Medicine, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yun Qiao; Chuan-Ju Liu, Email qlyyqy@163.com; Chuanju.Liu@nyulangone.orgObjective: This study is aimed to determine the metabolomic effects of the hybrid medicine formula Yi-Qi-Bu-Shen (YQBS) on the neurotransmitter aspects of cognitive impairment in diabetic rats.Methods: In the current study, streptozotocin (STZ) was used to induce diabetic animal model in male Sprague Dawley (SD) rats. After successful establishment of diabetic SD rats’ model, age-matched healthy SD rats and diabetic SD rats were treated with low and high doses of YQBS, and then tested for learning memory ability and analyzed for pathological changes. In addition, neurotransmitter metabolic changes in hippocampal subdivisions of rats from different treated groups were analyzed using liquid chromatography-mass spectrometry (LC-MS) technique.Results: YQBS could significantly improve memory-cognitive impairment in diabetic rats as evidenced by the shortening of latency to target and the reduction of latency first entrance to target. Moreover, YQBS also improved the pathological alterations in the hippocampal region in the brains of diabetic rats. Metabolomic analysis showed that the expression of noradrenaline hydrochloride was down-regulated and the expressions of levodopa and 5-hydroxytryptophan were up-regulated in the hippocampal tissues of diabetic rats treated with YQBS.Conclusion: These findings demonstrate that YQBS has protective effects against diabetic cognitive dysfunction, which might act through alteration in tyrosine and tryptophan metabolism.Keywords: diabetes mellitus, cognitive dysfunction, Yi-Qi-Bu-Shen, hippocampus, metabolomics

Mutations in GBA1 , encoding glucocerebrosidase (GCase), cause Gaucher disease (GD) and are also genetic risks in developing Parkinson’s disease (PD). Currently, the approved therapies are only effective for directly treating visceral symptoms, but not for primary neuronopathic involvement in GD (nGD). Progranulin (PGRN), encoded by GRN , is a novel modifier of GCase, but the impact of PGRN in GBA1 mutation-associated pathologies in vivo remains unknown. Herein, Grn −/− mice crossed into Gba 9v/9v mice, a Gba1 mutant line homozygous for the Gba1 D409V mutation, generating Grn −/− Gba 9v/9v (PG9V) mice. PG9V mice exhibited neurobehavioral deficits, early onset, and more severe GD phenotypes compared to Grn −/− and Gba 9v/9v mice. Moreover, PG9V mice also displayed PD-like phenotype. Mechanistic analysis revealed that PGRN deficiency caused severe neuroinflammation with microgliosis and astrogliosis, along with impaired autophagy associated with the Gba1 mutation. A PGRN-derived peptide, termed ND7, ameliorated the disease phenotype in GD patient fibroblasts ex vivo . Unexpectedly, ND7 penetrated the blood-brain barrier (BBB) and effectively ameliorated the nGD manifestations and PD pathology in Gba 9v/null and PG9V mice. Collectively, this study not only provides the first line of in vivo but also ex vivo evidence demonstrating the crucial role of PGRN in GBA1/Gba1 mutation-related pathologies, as well as a clinically relevant mouse model for mechanistic and potential therapeutics studies for nGD and PD. Importantly, a BBB penetrant PGRN-derived biologic was developed that may provide treatment for rare lysosomal storage diseases and common neurodegenerative disorders, particularly nGD and PD.

Progranulin (PGRN) is a key regulator of lysosomes, and its deficiency has been linked to various lysosomal storage diseases (LSDs), including Gaucher disease (GD), one of the most common LSD. Here, we report that PGRN plays a previously unrecognized role in autophagy within the context of GD. PGRN deficiency is associated with the accumulation of LC3-II and p62 in autophagosomes of GD animal model and patient fibroblasts, resulting from the impaired fusion of autophagosomes and lysosomes. PGRN physically interacted with Rab2, a critical molecule in autophagosome-lysosome fusion. Additionally, a fragment of PGRN containing the Grn E domain was required and sufficient for binding to Rab2. Furthermore, this fragment significantly ameliorated PGRN deficiency-associated impairment of autophagosome-lysosome fusion and autophagic flux. These findings not only demonstrate that PGRN is a crucial mediator of autophagosome-lysosome fusion but also provide new evidence indicating PGRN's candidacy as a molecular target for modulating autophagy in GD and other LSDs in general. KEY MESSAGES : PGRN acts as a crucial factor involved in autophagosome-lysosome fusion in GD. PGRN physically interacts with Rab2, a molecule in autophagosome-lysosome fusion. A 15-kDa C-terminal fragment of PGRN is required and sufficient for binding to Rab2. This PGRN derivative ameliorates PGRN deficiency-associated impairment of autophagy. This study provides new insights into autophagy and may develop novel therapy for GD.

The mammalian focal adhesion proteins Pinch1/2 activate integrins and promote cell–extracellular matrix adhesion and migration; however, their roles in adipose tissue and metabolism are unclear. Here we find that high-fat diet (HFD) feeding dramatically increases expression of Pinch1/2 proteins in white adipose tissue (WAT) in mice. Furthermore, expression of Pinch1 is largely upregulated in WAT in leptin-deficient ob/ob type 2 diabetic mice and obese humans. While mice with loss of Pinch1 in adipocytes or global Pinch2 do not display any notable phenotypes, deleting Pinch1 in adipocytes and Pinch2 globally significantly decreases body weight and WAT mass, but not brown adipose tissue mass, in HFD-fed, but not normal chow diet–fed, mice. Pinch loss ameliorates HFD-induced glucose intolerance and fatty liver. After HFD challenge, Pinch loss slightly but significantly accelerates energy expenditure. While Pinch loss decreases adipocyte size and alters adipocyte size distribution, it greatly accelerates cell apoptosis primarily in epididymal WAT and to a lesser extent in subcutaneous WAT. In vitro studies demonstrate that Pinch loss accelerates adipocyte apoptosis by activating the Bim/Caspase-8 pathway. In vivo, genetic ablation of Caspase-8 expression in adipocytes essentially abolishes the ameliorating effects of Pinch deficiency on obesity, glucose intolerance, and fatty liver in mice. Thus, we demonstrate a previously unknown function of Pinch in control of adipose mass, glucose, and fat metabolism via modulation of adipocyte apoptosis. We may define a novel target for the prevention and treatment of metabolic diseases, such as obesity and diabetes.

Balancing the process of bone formation and resorption is important in the maintenance of healthy bone. Therefore, the discovery of novel factors that can regulate bone metabolism remains needed. Irisin is a newly identified hormone‐like peptide. Recent studies have reported the involvement of irisin in many physiological and pathological conditions with bone mineral density changes, including osteopenia and osteoporotic fractures. In this study, we generated the first line of Osx‐Cre:FNDC5/irisin KO mice, in which FNDC5/irisin was specifically deleted in the osteoblast lineage. Gene and protein expressions of irisin were remarkably decreased in bones but no significant differences in other tissues were observed in knockout mice. FNDC5/irisin deficient mice showed a lower bone density and significantly delayed bone development and mineralization from early‐stage to adulthood. Our phenotypical analysis exhibited decreased osteoblast‐related gene expression and increased osteoclast‐related gene expression in bone tissues, and reduced adipose tissue browning due to bone‐born irisin deletion. By harvesting and culturing MSCs from the knockout mice, we found that osteoblastogenesis was inhibited and osteoclastogenesis was increased. By using irisin stimulated wildtype primary cells as a gain‐of‐function model, we further revealed the effects and mechanisms of irisin on promoting osteogenesis and inhibiting osteoclastogenesis in vitro. In addition, positive effects of exercise, including bone strength enhancement and body weight loss were remarkably weakened due to irisin deficiency. Interestingly, these changes can be rescued by supplemental administration of recombinant irisin during exercise. Our study indicates that irisin plays an important role in bone metabolism and the crosstalk between bone and adipose tissue. Irisin represents a potential molecule for the prevention and treatment of bone metabolic diseases.

Background and Aims: Recent studies have unveiled an association between ADAMTS7 gene variation and coronary artery disease (CAD) caused by atherosclerosis. We investigated if the ADAMTS7 Serine214-to-Proline substitution arising from a CAD-associated variant affected angiogenesis, since neovascularization plays an important role in atherosclerosis. Methods and Results: ADAMTS7 knockdown in vascular endothelial cells (ECs) attenuated their angiogenesis potential, whereas augmented ADAMTS7-Ser214 expression had the opposite effect, leading to increased ECs migratory and tube formation ability. Proteomics analysis showed an increase in thrombospondin-1, a reported angiogenesis inhibitor, in culture media conditioned by ECs with ADAMTS7 knockdown and a decrease of thrombospondin-1 in media conditioned by ECs with ADAMTS7-Ser214 overexpression. Cleavage assay indicated that ADAMTS7 possessed thrombospondin-1 degrading activity, which was reduced by the Ser214-to-Pro substitution. The pro-angiogenic effect of ADAMTS7-Ser214 diminished in the presence of a thrombospondin-1 blocking antibody. Conclusions: The ADAMTS7 Ser217-to-Pro substitution as a result of ADAMTS7 polymorphism affects thrombospondin-1 degradation, thereby promoting atherogenesis through increased EC migration and tube formation.

Troxerutin (TXR) is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit the antiarthritic properties of TXR using an adjuvant-induced arthritic (AIA) rat model. AIA-induced rats showed the highest arthritis score at the disease onset and by oral administration of TXR (50, 100, and 200 mg/kg body weight), reduced to basal level in a dose-dependent manner. Isobaric tags for relative and absolute quantitative (iTRAQ) proteomics tool were employed to identify deregulated joint homogenate proteins in AIA and TXR-treated rats to decipher the probable mechanism of TXR action in arthritis. iTRAQ analysis identified a set of 434 proteins with 65 deregulated proteins (log2 case/control≥1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from the healthy ones were validated using Western blot analysis. The Western blot data corroborated proteomics findings. In silico protein–protein interaction study of tissue-proteome revealed that complement component 9 (C9), the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, get perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an antiarthritic agent in the AIA model and after additional investigation, its arthritic ameliorating properties could be exploited for clinical usability.

Inflammatory Bowel Disease (IBD) is an autoimmune condition with complicated pathology and diverse clinical signs. TNFα is believed to play a crucial role in the pathogenesis of IBD. We recently identified fexofenadine, a well-known antagonist of histamine H1 receptor, as a novel inhibitor of TNFα signaling. Additionally, cytosolic phospholipase A2 (cPLA2) was isolated as a binding target of fexofenadine, and fexofenadine-mediated anti-TNF activity relied on cPLA2 in vitro. The objective of this study is to determine whether fexofenadine is therapeutic against chemically-induced murine IBD model and whether cPLA2 and/or histamine H1 receptor is important for fexofenadine’s anti-inflammatory activity in vivo by leveraging various genetically modified mice and chemically induced murine IBD models. Both dextran sulfate sodium- and 2, 4, 6-trinitrobenzene sulfonic acid-induced murine IBD models revealed that orally delivered fexofenadine was therapeutic against IBD, evidenced by mitigated clinical symptoms, decreased secretions of the proinflammatory cytokine IL-6 and IL-1β, lowered intestinal inflammation, and reduced p-p65 and p-IĸBα. Intriguingly, Fexofenadine-mediated protective effects against IBD were lost in cPLA2 deficient mice but not in histamine H1 receptor-deficient mice. Collectively, these findings demonstrate the therapeutic effects of over-the-counter drug Fexofenadine in treating DSS-induced IBD murine and provide first in vivo evidence showing that cPLA2 is required for fexofenadine’s therapeutic effects in murine IBD model and probably other inflammatory and autoimmune diseases as well.

ObjectivesDysregulated chondrocyte metabolism is closely associated with the pathogenesis of osteoarthritis (OA). Suppressing chondrocyte catabolism to restore cartilage homeostasis has been extensively explored, whereas far less effort has been invested toward enhancing chondrocyte anabolism. This study aimed to repurpose clinically approved drugs as potential stimulators of chondrocyte anabolism in treating OA.MethodsScreening of a Food and Drug Administration-approved drug library; Assays for examining the chondroprotective effects of digoxin in vitro; Assays for defining the therapeutic effects of digoxin using a surgically-induced OA model; A propensity-score matched cohort study using The Health Improvement Network to examine the relationship between digoxin use and the risk of joint OA-associated replacement among patients with atrial fibrillation; identification and characterisation of the binding of digoxin to low-density lipoprotein receptor-related protein 4 (LRP4); various assays, including use of CRISPR-Cas9 genome editing to delete LRP4 in human chondrocytes, for examining the dependence on LRP4 of digoxin regulation of chondrocytes.ResultsSerial screenings led to the identification of ouabain and digoxin as stimulators of chondrocyte differentiation and anabolism. Ouabain and digoxin protected against OA and relieved OA-associated pain. The cohort study of 56 794 patients revealed that digoxin use was associated with reduced risk of OA-associated joint replacement. LRP4 was isolated as a novel target of digoxin, and deletion of LRP4 abolished digoxin’s regulations of chondrocytes.ConclusionsThese findings not only provide new insights into the understanding of digoxin’s chondroprotective action and underlying mechanisms, but also present new evidence for repurposing digoxin for OA.

ObjectivesThis study aims to investigate the role and mechanism of FGFR3 in macrophages and their biological effects on the pathology of arthritis.MethodsMice with conditional knockout of FGFR3 in myeloid cells (R3cKO) were generated. Gait behaviours of the mice were monitored at different ages. Spontaneous synovial joint destruction was evaluated by digital radiographic imaging and μCT analysis; changes of articular cartilage and synovitis were determined by histological analysis. The recruitment of macrophages in the synovium was examined by immunostaining and monocyte trafficking assay. RNA-seq analysis, Western blotting and chemotaxis experiment were performed on control and FGFR3-deficient macrophages. The peripheral blood from non-osteoarthritis (OA) donors and patients with OA were analysed. Mice were treated with neutralising antibody against CXCR7 to investigate the role of CXCR7 in arthritis.ResultsR3cKO mice but not control mice developed spontaneous cartilage destruction in multiple synovial joints at the age of 13 months. Moreover, the synovitis and macrophage accumulation were observed in the joints of 9-month-old R3cKO mice when the articular cartilage was not grossly destructed. FGFR3 deficiency in myeloid cells also aggravated joint destruction in DMM mouse model. Mechanically, FGFR3 deficiency promoted macrophage chemotaxis partly through activation of NF-κB/CXCR7 pathway. Inhibition of CXCR7 could significantly reverse FGFR3-deficiency-enhanced macrophage chemotaxis and the arthritic phenotype in R3cKO mice.ConclusionsOur study identifies the role of FGFR3 in synovial macrophage recruitment and synovitis, which provides a new insight into the pathological mechanisms of inflammation-related arthritis.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by insulin deficiency, and patients with diabetes have an increased risk of bone fracture and significantly impaired fracture healing. Proinflammatory cytokine tumor necrosis factor-alpha is significantly upregulated in diabetic fractures and is believed to underlie delayed fracture healing commonly observed in diabetes. Our previous genetic screen for the binding partners of progranulin (PGRN), a growth factor-like molecule that induces chondrogenesis, led to the identification of tumor necrosis factor receptors (TNFRs) as the PGRN-binding receptors. In this study, we employed several in vivo models to ascertain whether PGRN has therapeutic effects in diabetic fracture healing. Here, we report that deletion of PGRN significantly delayed bone fracture healing and aggravated inflammation in the fracture models of mice with T1DM. In contrast, recombinant PGRN effectively promoted diabetic fracture healing by inhibiting inflammation and enhancing chondrogenesis. In addition, both TNFR1 proinflammatory and TNFR2 anti-inflammatory signaling pathways are involved in PGRN-stimulated diabetic fracture healing. Collectively, these findings illuminate a novel understanding concerning the role of PGRN in diabetic fracture healing and may have an application in the development of novel therapeutic intervention strategies for diabetic and other types of impaired fracture healing.

Progranulin (PGRN), a widely expressed glycoprotein with pleiotropic function, has been linked to a host of physiological processes and diverse pathological states. A series of contemporary preclinical disease models and clinical trials have evaluated various therapeutic strategies targeting PGRN, highlighting PGRN as a promising therapeutic target. Herein we summarize available knowledge of PGRN targeting in various kinds of diseases, including common neurological diseases, inflammatory autoimmune diseases, cancer, tissue repair, and rare lysosomal storage diseases, with a focus on the functional domain-oriented drug development strategies. In particular, we emphasize the role of extracellular PGRN as a non-conventional, extracellular matrix bound, growth factor-like conductor orchestrating multiple membrane receptors and intracellular PGRN as a chaperone/co-chaperone that mediates the folding and traffic of its various binding partners.

Esculetin is a coumarin compound, which belongs to the class of benzopyrone enriched in various plants such as Sonchus grandifolius, Aesculus turbinata, etc. Free radicals lead to the development of oxidative stress causing inflammation, arthritis, cancer, diabetes, fatty liver disease, etc. These further reduce the efficacy of anticancer drugs, activate inflammatory signaling pathways, degrade joints and cartilage, and disrupt the glycemic index and normal function of liver enzymes. For instance, the current treatment modalities used in arthritis such as non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatoid drugs, and lipoxygenase inhibitors present limited efficacy and adverse effects. Thus, there is a constant need to find newer and safer alternatives. Esculetin has an immense antioxidative potential thereby alleviating arthritis, diabetes, malignancies, and hepatic disorders. Structurally, esculetin contains two hydroxyl groups, which enhance its ability to function as an antioxidant by inhibiting oxidative stress in pathological conditions. Leukotriene B4 synthesis, NF-κB and MPAK pathway activation, and inflammatory cytokine production are the main causes of bone and joint deterioration in arthritis, whereas esculetin treatment reverses these factors and relieves the disease condition. In contrast, lipid peroxidation caused by upregulation of TGF-β-mediated expression and dysfunction of antioxidant enzymes is inhibited by esculetin therapy, thus reducing liver fibrosis by acting on the PI3K/FoxO1 pathway. Therefore, targeting NF-κB, pro-inflammatory cytokines, TGF-β and oxidative stress may be a therapeutic strategy to alleviate arthritis and liver fibrosis.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. We have successfully developed protein block polymers that consist of elastin like polypeptide (E) and the coiled-coil domain of cartilage oligomeric matrix protein (C). Here, we demonstrate that E5C, a construct consisting of five repeats of E and a single domain of C, is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin (PGRN), provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C hydrogels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the PGRN-derivative Atsttrin, for prevention of OA onset and progression.

Progranulin (PGRN) is a key regulator of lysosome and its deficiency has been linked to various lysosomal storage diseases (LSDs), including Gaucher disease (GD), one of the most common LSD. Here, we report that PGRN plays a previously unrecognized role in autophagy within the context of GD. PGRN deficiency associated with accumulation of LC3-II and p62 in autophagosomes of GD animal model and patient fibroblasts, resulting from impaired fusion of autophagosomes and lysosomes. PGRN physically interacted with Rab2, a critical molecule in autophagosome-lysosome fusion. Additionally, a fragment of PGRN containing the Grn E domain was required and sufficient for binding to Rab2. Further, this fragment significantly ameliorated PGRN-deficiency associated impairment of autophagosome-lysosome fusion and autophagic flux. These findings not only demonstrate that PGRN is a crucial mediator of autophagosome-lysosome fusion, but also provide new evidence indicating PGRN’s candidacy as a molecular target for modulating autophagy in GD and other LSDs in general.

OBJECTIVES: Type 2 diabetes mellitus (T2DM) significantly increases bone fragility and fracture risk. Progranulin (PGRN) promotes bone fracture healing in both physiological and type 1 diabetic conditions. The present study aimed to investigate the role of PGRN in T2DM bone fracture healing. METHODS: MKR (with FVB genetic background) mice were used as T2DM model. Drill-hole and Bonnarens and Einhorn models were established to investigate the role of PGRN in T2DM fracture healing in vivo. Primary bone marrow cells were isolated for molecular and signaling studies, and reverse transcriptase-polymerase chain reaction, immunohistochemical staining, and western blotting were performed to assess PGRN effects in vitro. RESULTS: PGRN mRNA and protein expression were upregulated in T2DM. Local administration of recombinant PGRN effectively promoted T2DM bone fracture healing in vivo. Additionally, PGRN could induce anabolic metabolism during endochondral ossification through TNFR2-Akt and Erk1/2 pathways. Furthermore, PGRN showed anti-inflammatory activity in the T2DM bone regeneration process. CONCLUSION: These findings suggest that local administration of exogenous PGRN may represent an alternative strategy to support bone regeneration in patients with T2DM. Additionally, PGRN might also hold therapeutic potential for other TNFR-related metabolic disorders.

TNFα/TNFR signaling plays a critical role in the pathogenesis of various inflammatory and autoimmune diseases, and anti-TNFα therapies have been accepted as the effective approaches for treating several autoimmune diseases. Progranulin (PGRN), a multi-faced growth factor-like molecule, directly binds to TNFR1 and TNFR2, particularly to the latter with higher affinity than TNFα. PGRN derivative Atsttrin is composed of three TNFR-binding domain of PGRN and exhibits even better therapeutic effects than PGRN in several inflammatory disease models, including collagen-induced arthritis. Herein we describe the detailed methodology of using (1) ELISA-based solid phase protein-protein interaction assay to demonstrate the direct binding of Atsttrin to TNFR2 and its inhibition of TNFα/TNFR2 interaction; and (2) tartrate-resistant acid phosphatase (TRAP) staining of in vitro osteoclastogenesis to reveal the cell-based anti-TNFα activity of Atsttrin. Using the protocol described here, the investigators should be able to reproducibly detect the physical inhibition of TNFα binding to TNFR and the functional inhibition of TNFα activity by Atsttrin and various kinds of TNF inhibitors.

TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical co-purification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3e as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3e was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3e resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3e signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPβ activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3e as a previously-unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3e anti-inflammatory pathway.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E(5)C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E(5)C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E(5)C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E(5)C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Tay-Sachs disease (TSD) is a lethal lysosomal storage disease (LSD) caused by mutations in the HexA gene, which can lead to deficiency of β-hexosaminidase A (HexA) activity and consequent accumulation of its substrate, GM2 ganglioside. Recent reports that progranulin (PGRN) functions as a chaperone of lysosomal enzymes and its deficiency is associated with LSDs, including Gaucher disease and neuronal ceroid lipofuscinosis, prompted us to screen the effects of recombinant PGRN on lysosomal storage in fibroblasts from 11 patients affected by various LSDs, which led to the isolation of TSD in which PGRN demonstrated the best effects in reducing lysosomal storage. Subsequent in vivo studies revealed significant GM2 accumulation and the existence of typical TSD cells containing zebra bodies in both aged and ovalbumin-challenged adult PGRN-deficient mice. In addition, HexA, but not HexB, was aggregated in PGRN-deficient cells. Furthermore, recombinant PGRN significantly reduced GM2 accumulation and lysosomal storage in these animal models. Mechanistic studies indicated that PGRN bound to HexA through granulins G and E domain and increased the enzymatic activity and lysosomal delivery of HexA. More importantly, Pcgin, an engineered PGRN derivative bearing the granulin E domain, also effectively bound to HexA and reduced the GM2 accumulation. Collectively, these studies not only provide new insights into the pathogenesis of TSD but may also have implications for developing PGRN-based therapy for this life-threatening disorder. KEY MESSAGES: GM2 accumulation and the existence of typical TSD cells containing zebra bodies are detected in both aged and ovalbumin-challenged adult PGRN deficient mice. Recombinant PGRN significantly reduces GM2 accumulation and lysosomal storage both in vivo and in vitro, which works through increasing the expression and lysosomal delivery of HexA. Pcgin, an engineered PGRN derivative bearing the granulin E domain, also effectively binds to to HexA and reduces GM2 accumulation.

p204, a murine member of the interferon-inducible p200 protein family, and its human analogue, IFI16, have been shown to function as tumor suppressors in vitro, but the molecular events involved, in particular in vivo, remain unclear. Herein we induced the Lewis Lung carcinoma (LLC) murine model of human lung cancer in p204 null mice (KO) and their control littermates (WT). We compared the transcriptome in spleen from WT and p204 KO mice using a high-throughput RNA-sequencing array. A total 30.02 Gb of clean data were obtained, and overall Q30% was greater than 90.54%. More than 75% of clean data from 12 transcriptome samples were mapped to exons. The results showed that only 11 genes exhibited altered expression in untreated p204 KO mice relative to untreated WT mice, while 393 altered genes were identified in tumor-bearing p204 KO mice when compared with tumor-bearing WT mice. Further differentially expressed gene cluster and gene ontology consortium classification revealed that numerous cytokines and their receptors, chemoattractant molecules, and adhesion molecules were significantly induced in p204 KO mice. This study provides novel insights to the p204 network in anti-tumor immune response and also presents a foundation for future work concerning p204-mediated gene expressions and pathways.

p204, a murine member of an interferon-inducible p200 family, was reported to recognize intracellular viral and bacterial DNAs, however, its role in the innate immunity in vivo remains unknown due to the lack of p204-deficient animal models. In this study we first generated the p204−/− mice. Unexpectedly, p204 deficiency led to significant defect in extracellular LPS signaling in macrophages, as demonstrated by dramatic reductions of LPS-mediated IFN-β and pro-inflammatory cytokines. The serum levels of IFN-β and pro-inflammatory cytokines were also significantly reduced in p204−/− mice following LPS challenge. In addition, p204−/− mice were resistant to LPS-induced shock. LPS-activated NF-ĸB and IRF-3 pathways were all defective in p204-deficient macrophages. p204 binds to TLR4 through its Pyrin domain, and it is required for the dimerization of TLR4 following LPS-challenge. Collectively, p204 is a critical component of canonical LPS-TLR4 signaling pathway, and these studies also suggest that p204 could be a potential target to prevent and treat inflammatory and infectious diseases. Keywords: p204, LPS, TLR4, IFN-β, Inflammatory responses, Macrophages

Autoimmune disease encompasses an array of conditions with a variety of presentations and the involvement of multiple organs. Though the etiologies of many autoimmune conditions are unclear, uncontrolled inflammatory immune response is believed to be a major cause of disease development and progression. Progranulin (PGRN), an anti-inflammatory molecule with therapeutic effect in inflammatory arthritis, was identified as an endogenous antagonist of TNFα by competitively binding to TNFR.. PGRN exerts its anti-inflammatory activity through multiple pathways, including induction of Treg differentiation and IL-10 expression and inhibition of chemokine release from macrophages. In addition, the protective role of PGRN has also been demonstrated in osteoarthritis, inflammatory bowel disease, and psoriasis. Intriguingly, PGRN was reported to contribute to development of insulin resistance in high-fat diet induced diabetes. Emerging evidences indicate that PGRN may also be associated with various autoimmune diseases, including systemic lupus erythematous, systemic sclerosis, multiple sclerosis and Sjogren’s syndrome. This review summarizes recent studies of PGRN as a novel target molecule in the field of autoimmune disease, and provides updated information to inspire future studies.