Induced TRIM21 ISGylation by IFN-β enhances p62 ubiquitination to prevent its autophagosome targeting

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-β also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-β.Subject terms: Proteins, Autophagy, Innate immunity, Post-translational modifications     

The Role of Toll-Like Receptor 9 in Chronic Stress-Induced Apoptosis in Macrophage

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.     

Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

Tumor necrosis factor alpha (TNF-α) plays a role in apoptosis and proliferation in multiple types of cells, and defects in TNF-α-induced apoptosis are associated with various autoimmune diseases. Here, we show that TRIM27, a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains, positively regulates TNF-α-induced apoptosis. Trim27-deficient mice are resistant to TNF-α–d-galactosamine-induced hepatocyte apoptosis. Trim27-deficient mouse embryonic fibroblasts (MEFs) are also resistant to TNF-α–cycloheximide-induced apoptosis. TRIM27 forms a complex with and ubiquitinates the ubiquitin-specific protease USP7, which deubiquitinates receptor-interacting protein 1 (RIP1), resulting in the positive regulation of TNF-α-induced apoptosis. Our findings indicate that the ubiquitination-deubiquitination cascade mediated by the TRIM27-USP7 complex plays an important role in TNF-α-induced apoptosis.     

TRIM22 activates NF-κB signaling in glioblastoma by accelerating the degradation of IκBα

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.Subject terms: CNS cancer, Oncogenes, Ubiquitin ligases     

PLEKHO2 inhibits TNFα-induced cell death by suppressing RIPK1 activation

Receptor interaction protein kinase 1 (RIPK1) plays a diverse role in tumor necrosis factor α (TNFα) signalings. The ubiquitination of RIPK1 is essential for NF-κB activation, whereas its kinase activity promotes apoptosis and necroptosis. However, the mechanisms underlying have not been fully illuminated. Here we report that PH domain-containing family O member 2 (PLEKHO2) inhibits RIPK1-dependent cell death and is necessary for NF-κB activation in response to TNFα. Cells of PLKEHO2 deficiency are more susceptible to TNF-α induced apoptosis and necroptosis with increased RIPK1 activation, which is consistent with the observation that the susceptibility of PLEKHO2−/− cells is effectively prevented by treatment of RIPK1 kinase inhibitor. Moreover, PLEKHO2 deficient cells exhibit compromised RIPK1 ubiquitination and NF-κB activation in response to TNFα. Ultimately, PLEKHO2-deficient mice display greatly increased hepatotoxicity and lethality after TNFα-induced hepatitis. In summary, our study revealed that PLEKHO2 is a novel inhibitor of apoptosis and necroptosis, which plays a key role in regulating RIPK1 ubiquitination and activationSubject terms: Apoptosis, Ubiquitylation     

Macrophage activation state determines the response to rhinovirus infection in a mouse model of allergic asthma

Background

The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.

Methods

Eight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate.

Results

In contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages.

Conclusions

OVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.     

Transient receptor potential melastatin 2–antisense RNA is overexpresed in osteosarcoma and regulates cell proliferation and apoptosis

Accumulating evidence suggested that transient receptor potential melastatin 2–antisense RNA (TRPM2-AS) played crucial roles in the progression of human cancers. However, the role of TRPM2-AS was still unknown in osteosarcoma. The aim of this study was to explore the clinical significance of TRPM2-AS in osteosarcoma patients, and determine the role of TRPM2-AS on osteosarcoma cell proliferation and apoptosis. In our results, we identified a novel oncogenic long noncoding RNA TRPM2-AS, which was overexpressed in osteosarcoma tissues and cells, and correlated with advanced Enneking stage, large tumor size and high histological grade in osteosarcoma cases. Survival analysis indicated that osteosarcoma patients with high TRPM2-AS expression had an obviously shorter overall survival time than those with low TRPM2-AS expression. Loss-of-function studies suggested that suppression of TRPM2-AS expression inhibited osteosarcoma cell proliferation and induced cell apoptosis through upregulating cleaved caspase-3 and cleaved caspase-9 expression. In conclusion, TRPM2-AS acts as an oncogenic long noncoding RNA and predicts poor prognosis in osteosarcoma.     

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.     

Local Immunosuppressive Microenvironment Enhances Migration of Melanoma Cells to Lungs in DJ-1 Knockout Mice

DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1β expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×10⁴) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1β and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1β) neutralizing antibody to see whether IL-1β is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1β to explore underlying molecular mechanisms. Our results showed that IL-1β enhanced survival and colony formation of cultured melanoma cells, and that IL-1β levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1β correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1β neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1β plays an important role in promoting the cancer migration.     

Wnt5a Promotes Inflammatory Responses via Nuclear Factor κB (NF-κB) and Mitogen-activated Protein Kinase (MAPK) Pathways in Human Dental Pulp Cells

Wnt5a has been found recently to be involved in inflammation regulation through a mechanism that remains unclear. Immunohistochemical staining of infected human dental pulp and tissue from experimental dental pulpitis in rats showed that Wnt5a levels were increased. In vitro, Wnt5a was increased 8-fold in human dental pulp cells (HDPCs) after TNF-α stimulation compared with control cells. We then investigated the role of Wnt5a in HDPCs. In the presence of TNF-α, Wnt5a further increased the production of cytokines/chemokines, whereas Wnt5a knockdown markedly reduced cytokine/chemokine production induced by TNF-α. In addition, in HDPCs, Wnt5a efficiently induced cytokine/chemokine expression and, in particular, expression of IL-8 (14.5-fold) and CCL2 (25.5-fold), as assessed by a Luminex assay. The cytokine subsets regulated by Wnt5a overlap partially with those induced by TNF-α. However, no TNF-α and IL-1β was detected after Wnt5a treatment. We then found that Wnt5a alone and the supernatants of Wnt5a-treated HDPCs significantly increased macrophage migration, which supports a role for Wnt5a in macrophage recruitment and as an inflammatory mediator in human dental pulp inflammation. Finally, Wnt5a participates in dental pulp inflammation in a MAPK-dependent (p38-, JNK-, and ERK-dependent) and NF-κB-dependent manner. Our data suggest that Wnt5a, as an inflammatory mediator that drives the integration of cytokines and chemokines, acts downstream of TNF-α.     

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute in vitro exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.     

Morin,a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators,Intracellular ROS Levels and NF-κB Activation

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG₂), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.     

Glucose regulates expression of pro-inflammatory genes,IL-1β and IL-12, through a mechanism involving hexosamine biosynthesis pathway-dependent regulation of α-E catenin

High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose-induced increase in α-E catenin when hexosamine biosynthesis (HB) pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of HB pathway in this process. Then, we investigated the potential role of α-E catenin in glucose-induced macrophage polarisation. We find that the reduction in α-E catenin level using siRNA attenuates the glucose-induced changes of both IL-1β and IL-12 mRNA levels under LPS-stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via HB pathway and also can modulate the glucose-induced gene expression of inflammatory markers such as IL-1β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.     

Apparent effect of rabbit endogenous lentivirus type K acquisition on retrovirus restriction by lagomorph Trim5αs

To test the hypothesis that rabbit endogenous lentivirus type K (RELIK) could play a role in shaping the evolution of TRIM5α, the susceptibility of viruses containing the RELIK capsid (CA) to TRIM5 restriction was evaluated. RELIK CA-containing viruses were susceptible to the TRIM5αs from Old World monkeys but were unaffected by most ape or New World monkey factors. TRIM5αs from various lagomorph species were also isolated and tested for anti-retroviral activity. The TRIM5αs from both cottontail rabbit and pika restrict a range of retroviruses, including HIV-1, HIV-2, FIV, EIAV and N-MLV. TRIM5αs from the European and cottontail rabbit, which have previously been found to contain RELIK, also restricted RELIK CA-containing viruses, whereas a weaker restriction was observed with chimeric TRIM5α containing the B30.2 domain from the pika, which lacks RELIK. Taken together, these results could suggest that the pika had not been exposed to exogenous RELIK and that endogenized RELIK might exert a selective pressure on lagomorph TRIM5α.     

Nod-Like Receptor Protein-3 Inflammasome Plays an Important Role during Early Stages of Wound Healing

The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.     

IL-1β Production through the NLRP3 Inflammasome by Hepatic Macrophages Links Hepatitis C Virus Infection with Liver Inflammation and Disease

Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.