Sec-O-Glucosylhamaudol Inhibits RANKL-Induced Osteoclastogenesis by Repressing 5-LO and AKT/GSK3ß Signaling.

Sec-O-glucosylhamaudol (SOG), an active flavonoid compound derived from the root of Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., exhibits analgesic, anti-inflammatory, and high 5-lipoxygenase (5-LO) inhibitory effects. However, its effect on osteoclastogenesis was unclear. We demonstrated that SOG markedly attenuated RANKL-induced osteoclast formation, F-actin ring formation, and mineral resorption by reducing the induction of key transcription factors NFATc1, c-Fos, and their target genes such as TRAP, CTSK, and DC-STAMP during osteoclastogenesis. Western blotting showed that SOG significantly inhibited the phosphorylation of AKT and GSK3ß at the middle-late stage of osteoclastogenesis without altering calcineurin catalytic subunit protein phosphatase-2ß-Aα expression. Moreover, GSK3ß inhibitor SB415286 partially reversed SOG-induced inhibition of osteoclastogenesis, suggesting that SOG inhibits RANKL-induced osteoclastogenesis by activating GSK3ß, at least in part. 5-LO gene silencing by small interfering RNA in mouse bone marrow macrophages markedly reduced RANKL-induced osteoclastogenesis by inhibiting NFATc1. However, it did not affect the phosphorylation of AKT or GSK3ß, indicating that SOG exerts its inhibitory effects on osteoclastogenesis by suppressing both the independent 5-LO pathway and AKT-mediated GSK3ß inactivation. In support of this, SOG significantly improved bone destruction in a lipopolysaccharide-induced mouse model of bone loss. Taken together, these results suggest a potential therapeutic effect for SOG on osteoclast-related bone lysis disease.

Administration of Exogenous Progesterone Protects Against Brucella abortus Infection-Induced Inflammation in Pregnant Mice.

Progesterone has been recognized as essential for the establishment and maintenance of pregnancy, and is typically known as an immunosuppressive agent. However, its effects on mediating Brucella infection-induced inflammation have not been evaluated. Here we demonstrated that Brucella abortus infection inhibits progesterone levels in the pregnant mouse by suppressing the production of progesterone by placenta. Progesterone treatment significantly reduced the secretion of inflammatory cytokines in serum, macrophages, and trophoblasts of B. abortus-infected mice, leading to decreased placentitis and enhancing the pup viability. Mechanistically, this decreased inflammatory response results from inhibition of NF-kB activation by progesterone. Moreover, progesterone treatment suppresses B. abortus growth within trophoblasts associated with an inability of bacteria to escape the late endosome compartment in vitro. Collectively, our data illustrate that progesterone treatment might be useful therapeutically in protection against placentitis or abortion caused by B. abortus infection.

HMGB1 release from trophoblasts contributes to inflammation during Brucella melitensis infection.

Brucella melitensis infection causes acute necrotizing inflammation in pregnant animals; however, the pathophysiological mechanisms leading to placentitis are unknown. Here, we demonstrate that high-mobility group box 1 (HMGB1) acts as a mediator of placenta inflammation in B. melitensis-infected pregnant mice model. HMGB1 levels were increased in trophoblasts or placental explant during B. melitensis infection. Inhibition of HMGB1 activity with neutralising antibody significantly reduced the secretion of inflammatory cytokines in B. melitensis-infected trophoblasts or placenta, whereas administration of recombinant HMGB1 (rHMGB1) increased the inflammatory response. Mechanistically, this decreased inflammatory response results from inhibition of HMGB1 activity, which cause the suppression of both mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Moreover, neutralising antibody to HMGB1 prevented B. melitensis infection-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in trophoblasts. In contrast, in vitro stimulation of trophoblasts with rHMGB1 caused activation of NADPH oxidase and increased the production of ROS, which contributes to high bacterial burden within trophoblasts or placenta. In vivo, treatment with anti-HMGB1 antibody increases the number of Brucella survival within placenta in B. melitensis-infected pregnant mice but successfully reduced the severity of placentitis and abortion.

Inhibition of nucleolar stress response by Sirt1: A potential mechanism of acetylation-independent regulation of p53 accumulation.

The mammalian Sirt1 deacetylase is generally thought to be a nuclear protein, but some pilot studies have suggested that Sirt1 may also be involved in orchestrating nucleolar functions. Here, we show that nucleolar stress response is a ubiquitous cellular reaction that can be induced by different types of stress conditions, and Sirt1 is an endogenous suppressor of nucleolar stress response. Using stable isotope labeling by amino acids in cell culture approach, we have identified a physical interaction of between Sirt1 and the nucleolar protein nucleophosmin, and this protein-protein interaction appears to be necessary for Sirt1 inhibition on nucleolar stress, whereas the deacetylase activity of Sirt1 is not strictly required. Based on the reported prerequisite role of nucleolar stress response in stress-induced p53 protein accumulation, we have also provided evidence suggesting that Sirt1-mediated inhibition on nucleolar stress response may represent a novel mechanism by which Sirt1 can modulate intracellular p53 accumulation independent of lysine deacetylation. This process may represent an alternative mechanism by which Sirt1 regulates functions of the p53 pathway.

Tatarinan N inhibits osteoclast differentiation through attenuating NF-κB, MAPKs and Ca2+-dependent signaling.

Osteoclasts are multinucleated cells that originate from hemopoietic stem cells. Targeting over activated osteoclasts is thought to be an effective therapeutic approach to osteoporosis. In a previous study, we reported that Tatarinan O, a lignin-like compound, suppressed RANKL-induced osteoclastogenesis. In this study, we further examined the effects on osteoclast formation of three lignin-like compounds including Tatarinan N (TN), Tatarinan U (TU) and Tatarinan V (TV), all containing a common structure of asarone. We found that only TN suppressed RANKL-induced osteoclast differentiation, bone resorption pit formation and F-acting ring formation. TU and TV did not influence RANKL-induced osteoclastogenesis. We also found that TN dose-dependently inhibited the expression of osteoclastogenesis-associated genes, including TRAP, cathepsin K and MMP-9. Furthermore, we found that TN down-regulated the key transcription factor NFATc1 and c-Fos by preventing the activation of NF-κB and phosphorylation of MAPKs including ERK1/2 and p38 but not JNK. TN attenuated calcineurin expression via suppression of the Btk-PLCγ2 cascade and reduction of intracellular Ca2+, modulating NFATc1 activation. Taking together, our results indicated that TN might have therapeutic potential for osteoporosis.

DOK3 Modulates Bone Remodeling by Negatively Regulating Osteoclastogenesis and Positively Regulating Osteoblastogenesis.

Osteoclastogenesis is essential for bone remodeling and normal skeletal maintenance. Receptor activator of NF-κB ligand (RANKL) promotes osteoclast differentiation and function but requires costimulation of immunoreceptor tyrosine-based activation motif (ITAM)-coupled immunoreceptors. Triggering receptor expressed on myeloid cells-2 (TREM2) coupled to ITAM-adaptor protein DNAX activation protein 12kDA (DAP12) provides costimulation of intracellular calcium signaling during osteoclastogenesis. Previously, we found that downstream of kinase-3 (DOK3) physically associates with DAP12 to inhibit toll-like receptor (TLR)-induced inflammatory signaling in macrophages. However, whether and how DOK3 modulates DAP12-dependent osteoclastogenesis is unknown and the focus of this study. Bone microarchitecture and histology of sex- and age-matched wild-type (WT) and DOK3-deficient (DOK3-/- ) mice were evaluated. Male and female DOK3-/- mice have significantly reduced trabecular bone mass compared with WT mice with increased TRAP+ osteoclasts in vivo. In vitro, DOK3-/- bone marrow-derived macrophages (BMMs) have increased macrophage colony-stimulating factor (M-CSF)-induced proliferation and increased sensitivity to RANKL-induced osteoclastogenesis. Compared with WT, DOK3-/- osteoclasts are significantly larger with more nuclei and have increased resorptive capacity. Mechanistically, DOK3 limits osteoclastogenesis by inhibiting activation of Syk and ERK in response to RANKL and M-CSF. DOK3 is phosphorylated in a DAP12-dependent manner and associates with Grb2 and Cbl. Compared with DAP12-/- mice with high bone mass, DOK3- and DAP12- doubly deficient mice (DKO) have normalized bone mass, indicating that DOK3 also limits DAP12-independent osteoclastogenesis in vivo. In vitro osteoclasts derived from DKO mice are mononuclear with poor resorptive capacity similar to DAP12-/- osteoclasts. Histomorphometry reveals that DOK3-/- mice also have reduced osteoblast parameters. DOK3-/- osteoblasts have reduced in vitro osteoblastogenesis and increased osteoprotegerin (OPG) to RANKL expression ratio compared with WT osteoblasts. Co-culture of WT and DOK3-/- osteoblasts with pre-osteoclasts reveals a reduced capacity of DOK3-/- osteoblasts to support osteoclastogenesis. These data indicate that DOK3 regulates bone remodeling by negatively regulating M-CSF- and RANKL-mediated osteoclastogenesis and positively regulating osteoblastogenesis. © 2017 American Society for Bone and Mineral Research.

Tert-butylhydroquinone promotes angiogenesis and improves heart functions in rats after myocardial infarction.

BACKGROUND: Hypertension is an increased risk of heart failure and acute myocardial infarction (MI). Tert-butylhydroquinone (tBHQ), as an antioxidant, shows multiple cardioprotective actions including the reduction in blood pressure. The aim of this study was to investigate whether and how tBHQ improves heart functions in rats. METHODS: The MI model was established in WKY and spontaneously hypertensive rats (SHRs) by ligation of left anterior descending coronary artery. Akt phosphorylation was examined by western blot in human umbilical vein endothelial cells (HUVECs) or in rats. Angiogenesis was assessed by immunohistochemistry and immunofluorescence. Heart function was determined by echocardiography. RESULTS: tBHQ increased Akt phosphorylation, promoted cell proliferations and migrations in HUVECs, which were abolished by Akt inhibitor wortmannin. In SHRs following MI, administration of tBHQ significantly increased Akt phosphorylation, promoted angiogenesis, reduced infarct size, and improved heart functions after 14 postoperative days. Importantly, these in vivo effects of tBHQ were ablated by wortmannin in SHRs. CONCLUSION: tBHQ via Akt activation promotes ischemia-induced angiogenesis and improves heart functions in hypertensive rats. In perspectives, the application of tBHQ should be considered in patients with ischemic diseases such as MI and stroke.

MicroRNA-125b-5p suppresses Brucella abortus intracellular survival via control of A20 expression.

BACKGROUND: Brucella may establish chronic infection by regulating the expression of miRNAs. However, the role of miRNAs in modulating the intracellular growth of Brucella remains unclear. RESULTS: In this study, we show that Brucella. abortus infection leads to downregulation of miR-125b-5p in macrophages. We establish that miR-125b-5p targets A20, an inhibitor of the NF-kB activation. Additionally, expression of miR-125b-5p decreases A20 expression in B. abortus-infected macrophages and leads to NF-kB activation and increased production of TNFα. Furthermore, B. abortus survival is attenuated in the presence of miR-125b-5p. CONCLUSIONS: These results uncover a role for miR-125b-5p in the regulation of B. abortus intracellular survival via the control of A20 expression.

Tatarinan O, a lignin-like compound from the roots of Acorus tatarinowii Schott inhibits osteoclast differentiation through suppressing the expression of c-Fos and NFATc1.

Osteoclasts (OC) are large multinucleated cells derived from monocyte/macrophage precursors. Suppressing osteoclastogenesis is considered as an effective therapeutic approach to erosive bone disease. The root of Acorus tatarinowii Schott, a well-known traditional Chinese medicine was used to treat rheumatosis and other inflammatory disease. However, the effects of tatarinan O (TO), one of the lignin-like compounds isolated from the roots of Acorus tatarinowii Schott during bone development are still unclear. In the present study, we explored the effect of TO on RANKL-induced osteoclastogenesis in vitro. TO was found to suppress osteoclast differentiation from RANKL-stimulated mouse bone marrow macrophages (BMMs) without significant cytotoxicity. TO also dose-dependently suppressed bone resorption activity of mature osteoclasts. Additionally, TO apparently inhibited the expression of osteoclastic marker genes, such as MMP-9, Cts K and TRAP. Furthermore, our results showed that TO decreased RANKL-induced expression of c-Fos and NFATc1 without influencing NF-κB activation and MAPK phosphorylation. Hence, for the first time we revealed that TO dose-dependently inhibited osteoclastogenesis from RANKL-stimulated mouse BMMs via decreasing the expression of NFATc1 and c-Fos.

Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth.

UNLABELLED: AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKß, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE: Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production.

The Protective Effects of HJB-1, a Derivative of 17-Hydroxy-Jolkinolide B, on LPS-Induced Acute Distress Respiratory Syndrome Mice.

Acute respiratory distress syndrome (ARDS),which is inflammatory disorder of the lung, which is caused by pneumonia, aspiration of gastric contents, trauma and sepsis, results in widespread lung inflammation and increased pulmonary vascular permeability. Its pathogenesis is complicated and the mortality is high. Thus, there is a tremendous need for new therapies. We have reported that HJB-1, a 17-hydroxy-jolkinolide B derivative, exhibited strong anti-inflammatory effects in vitro. In this study, we investigated its impacts on LPS-induced ARDS mice. We found that HJB-1 significantly alleviated LPS-induced pulmonary histological alterations, inflammatory cells infiltration, lung edema, as well as the generation of inflammatory cytokines TNF-α, IL-1ß and IL-6 in BALF. In addition, HJB-1 markedly suppressed LPS-induced IκB-α degradation, nuclear accumulation of NF-κB p65 subunit and MAPK phosphorylation. These results suggested that HJB-1 improved LPS-induced ARDS by suppressing LPS-induced NF-κB and MAPK activation.

Calycosin Suppresses RANKL-Mediated Osteoclastogenesis through Inhibition of MAPKs and NF-κB.

Calycosin, an isoflavonoid phytoestrogen, isolated from Radix Astragali, was reported to possess anti-tumor, anti-inflammation, and osteogenic properties, but its impact on osteoclast differentiation remains unclear. In this study, we examined the effects of calycosin on osteoclastogenesis induced by RANKL. The results showed that calycosin significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). Calycosin also dose-dependently suppressed the formation of bone resorption pits by mature osteoclasts. In addition, the expression of osteoclatogenesis-related genes, including cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), and MMP-9, was significantly inhibited by calycosin. Furthermore, the results indicated that calycosin down-regulated the expression levels of NFATc1 and c-Fos through suppressing the activation of NF-κB and MAPKs. Our results indicate that calycosin has an inhibitory role in the bone loss by preventing osteoclast formation, as well as its bone resorptive activity. Therefore, calycosin may be useful as a therapeutic reagent for bone loss-associated diseases.

Proteomics Analysis of Cellular Proteins Co-Immunoprecipitated with Nucleoprotein of Influenza A Virus (H7N9).

Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

TRAF6-mediated degradation of DOK3 is required for production of IL-6 and TNFα in TLR9 signaling.

Our previous study showed that the downstream of kinase 3 (DOK3) is degraded during macrophage stimulation with CpG. However, the underlying mechanism and role in Toll-like receptor 9 (TLR9) signaling remains elusive. In this study, we demonstrate that CpG treatment leads to ubiquitin-mediated degradation of DOK3 via interaction with an E3 ligase TNFR-associated factor 6 (TRAF6). We also identified the 27th amino acid (lysine) of DOK3 is responsible for Ly48 polyubiquitination of DOK3. Furthermore, reintroduction of DOK3 (K27R) into DOK3-deficient macrophages abolishes DOK3 degradation induced by CpG and suppresses the production of IL-6 and TNFα. More importantly, our study uncovers a novel role of an E3 ligase TRAF6, namely, TRAF6 is also able to catalyse Lys 48 polyubiquitylation of target protein except for Lys 63 polyubiquitylation.

17-Hydroxy-jolkinolide A inhibits osteoclast differentiation through suppressing the activation of NF-κB and MAPKs.

Osteoclasts (OC) are bone-specific multinucleated giant cells (MNCs) derived from the monocyte/macrophage hematopoietic lineage cells. Inhibiting osteoclast formation is considered as an effective therapeutic approach for the treatment of the pathological bone loss. In this study, we investigated effects of 17-hydroxy-jolkinolide A (HJA), an ent-abietane diterpenoid isolated from the dried root of Euphorbia fischeriana, on osteoclastogenesis induced by RANKL. The results showed that HJA significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). HJA also prevented bone resorption by mature osteoclasts in a dose-dependent manner. In addition, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (Cts K) and MMP-9, was significantly inhibited by HJA. Furthermore, HJA also significantly inhibited RANKL-induced activation of NF-κB and phosphorylation of MAPK. Our results indicate that HJA has an inhibitory role in the bone loss by preventing osteoclast formation as well as its bone resorptive activity. Therefore, HJA may be useful as a therapeutic reagent for bone loss-associated diseases.

Berberine via suppression of transient receptor potential vanilloid 4 channel improves vascular stiffness in mice.

Berberine, as an alkaloid found in many Chinese herbs, improves vascular functions in patients with cardiovascular diseases. We determined the effects of berberine in hypertension and vascular ageing, and elucidated the underlying mechanisms. In isolated aortas, berberine dose-dependently elicited aortic relaxation. In cultured cells, berberine induced the relaxation of vascular smooth muscle cells (VSMCs). Overexpression of transient receptor potential vanilloid 4 (TRPV4) channel by genetic approaches abolished the berberine-induced reduction in intracellular Ca(2+) concentration in VSMCs and attenuated berberine-elicited vessel dilation in mice aortas. In deoxycorticosterone acetate (DOCA)-induced hypertensive model, treatment of mice with berberine or RN-1734, a pharmacological inhibitor of TRPV4, significantly decreased systemic blood pressure (BP) in control mice or mice infected with an adenovirus vector. However, berberine-induced effects of lowering BP were reversed by overexpressing TRPV4 in mice by infecting with adenovirus. Furthermore, long-term administration of berberine decreased mean BP and pulse BP, increased artery response to vasodilator and reduced vascular collagen content in aged mice deficient in apolipoprotein E (Apoe-KO), but not in Apoe-KO old mice with lentivirus-mediated overexpression of TRPV4 channel. In conclusion, berberine induces direct vasorelaxation to lower BP and reduces vascular stiffness in aged mice through suppression of TRPV4.

The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages.

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

A20 promotes Brucella intracellular growth via inhibition of macrophage cell death and activation.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of macrophage activation and apoptosis in tumor necrosis factor receptor1 (TNFR1) signaling pathway. Brucella infection can induce A20 expression in macrophages. Here, we hypothesize that A20 promotes Brucella intracellular growth via inhibition of activation and apoptosis of macrophages. To test this hypothesis, we stably incorporated mouse A20-shRNA into the RAW264.7 cells by lentiviral gene transfer to successfully knockdown A20. A20-deficient RAW264.7 cells were subsequently challenged with Brucella abortus and colony formation units (CFUs) of bacteria, TNFα production, NF-kB activation, macrophages apoptosis and cell death were evaluated. The A20 knockdown was shown to effectively promote B. abortus-stimulated TNFα release, NF-kB activation and macrophage cell death, which suppressed B. abortus intracellular replication. Unexpectedly, deficiency of A20 failed to lead to B. abortus-induced macrophage apoptosis. A20 deficiency coupled NF-kB inhibition promoted caspase-8 dependent B. abortus-induced macrophage apoptosis. These findings provide a novel mechanism by which Brucella intracellular growth within macrophages occurs through up-regulation of A20 thereby limiting activation and macrophages cell death.

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis.