Chronic mTOR inhibition in mice with rapamycin alters T,B, myeloid,and innate lymphoid cells and gut flora and prolongs life of immune‐deficient mice

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.     

Anti‐inflammaging effects of human alpha‐1 antitrypsin

Inflammaging plays an important role in most age‐related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha‐1 antitrypsin (hAAT) has immune‐regulatory, anti‐inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti‐inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT‐expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti‐aging effect of hAAT, we monitored the expression of aging‐associated genes and found that aging‐induced expressions of Relish (NF‐?B orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA‐seq analysis revealed that innate immunity genes regulated by NF‐kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti‐inflammaging effect in human cells, we treated X‐ray‐induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL‐6 and IL‐8, two major factors of senescence‐associated secretory phenotype. Consistent with results from Drosophila,RNA‐seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA‐approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging‐related diseases.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

Silencing of ATP2B1‐AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA‐mediated NF‐κB signalling pathway

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca²⁺ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.     

Advanced glycation end products‐related modulation of cathepsin L and NF‐κB signalling effectors in retinal pigment epithelium lead to augmented response to TNFα

The retinal pigment epithelium (RPE) plays a central role in neuroretinal homoeostasis throughout life. Altered proteolysis and inflammatory processes involving RPE contribute to the pathophysiology of age‐related macular degeneration (AMD), but the link between these remains elusive. We report for the first time the effect of advanced glycation end products (AGE)—known to accumulate on the ageing RPE's underlying Bruch's membrane in situ—on both key lysosomal cathepsins and NF‐κB signalling in RPE. Cathepsin L activity and NF‐κB effector levels decreased significantly following 2‐week AGE exposure. Chemical cathepsin L inhibition also decreased total p65 protein levels, indicating that AGE‐related change of NF‐κB effectors in RPE cells may be modulated by cathepsin L. However, upon TNFα stimulation, AGE‐exposed cells had significantly higher ratio of phospho‐p65(Ser536)/total p65 compared to non‐AGEd controls, with an even higher fold increase than in the presence of cathepsin L inhibition alone. Increased proportion of active p65 indicates an AGE‐related activation of NF‐κB signalling in a higher proportion of cells and/or an enhanced response to TNFα. Thus, NF‐κB signalling modulation in the AGEd environment, partially regulated via cathepsin L, is employed by RPE cells as a protective (para‐inflammatory) mechanism but renders them more responsive to pro‐inflammatory stimuli.     

Corilagin suppresses RANKL‐induced osteoclastogenesis and inhibits oestrogen deficiency‐induced bone loss via the NF‐κB and PI3K/AKT signalling pathways

Over‐activated osteoclastogenesis, which is initiated by inflammation, has been implicated in osteoporosis. Corilagin, a natural compound extracted from various medicinal herbaceous plants, such as Cinnamomum cassia, has antioxidant and anti‐inflammatory activities. We found that Corilagin suppressed osteoclast differentiation in a dose‐dependent manner, significantly decreased osteoclast‐related gene expression and impaired bone resorption by osteoclasts. Moreover, phosphorylation of members of the nuclear factor‐kappaB (NF‐κB) and PI3K/AKT signalling pathways was reduced by Corilagin. In a murine model of osteoporosis, Corilagin inhibited osteoclast functions in vivo and restored oestrogen deficiency‐induced bone loss. In conclusion, our findings suggested that Corilagin inhibited osteoclastogenesis by down‐regulating the NF‐κB and PI3K/AKT signalling pathways, thus showing its potential possibility for the treatment of osteoporosis.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

The ubiquitin‐editing enzyme A20 requires RNF11 to downregulate NF‐κB signalling

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor‐beta (TGF‐β) signalling. RNF11 has been proposed to regulate TGF‐β signalling by interacting with HECT‐ and SCF‐type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF‐κB and jun N‐terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)‐ and lipopolysaccharide (LPS)‐mediated NF‐κB and JNK signalling. RNF11 interacted with the NF‐κB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus‐dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF‐mediated NF‐κB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin‐editing protein complex that ensures transient activation of inflammatory signalling pathways.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Small RNAs induce the activation of the pro‐inflammatory TLR7 signaling pathway in aged rat kidney

We have recently reported that TLR‐related genes, including TLR7, are upregulated during aging. However, the role of TLR7 and its endogenous ligand in inflammation related to aging is not well defined. Here, we established that small RNAs trigger age‐related renal inflammation via TLR7 signaling pathway. We first investigated the expression changes of nine different TLRs in kidney of 6‐month‐old young rats and 20‐month‐old aged rats. The results revealed that the expression of TLR7 was the highest among nine TLRs in kidney of old rats compared to the young aged rats. Next, to assess the role of cellular RNA as a TLR7 ligand, we treated a renal tubular epithelial cell line with total RNA isolated from the kidney of young and old rats. The results showed that RNA isolated from old rats showed higher expression of TLR7, IL1β, and TNFα compared to that from young rats. Furthermore, RNA isolated from old rats induced IKKα/β/JNK/NF‐κB activation. To identify RNA that activates TLR7, we isolated small and large RNAs from old rat kidney and found that small RNAs increased TLR7 expression in cells. Finally, to investigate the local inflammatory response by small RNA, C57B/L6 mice were intraperitoneally injected with small RNAs isolated from young and old rats; thereby, RNA isolated from old rats induced higher inflammatory responses. Our study demonstrates that renal small RNAs from aged rats induce pro‐inflammatory processes via the activation of the TLR7/IKKα/β/JNK/NF‐κB signaling pathway, and highlights its causative role as a possible therapeutic target in age‐related chronic renal inflammation.     

Cell entry of bovine ephemeral fever virus requires activation of Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB pathways as well as Cox‐2‐mediated PGE2/EP receptor signalling to enhance clathrin‐mediated virus endocytosis

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin‐mediated and dynamin 2‐dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src‐JNK signalling to enhance its entry. Activation of these pathways by ultraviolet‐inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV‐induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3′‐dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox‐2‐catalysed prostaglandin E₂ (PGE₂) synthesis and induces expressions of G‐protein‐coupled E‐prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV‐induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox‐2‐mediated PGE₂/EP receptor signalling pathways, further enhancing Src‐JNK‐AP1 in a cAMP‐dependent manner and PI3K‐Akt‐NF‐κB in a cAMP‐independent manner. Accordingly, BEFV stimulates PGE₂/EP receptor signalling amplifying Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB pathways in an autocrine or paracrine fashion to enhance virus entry.