Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Transcriptional response of human articular chondrocytes treated with fibronectin fragments: an in vitro model of the osteoarthritis phenotype.

OBJECTIVE: Fibronectin is a matrix protein that is fragmented during cartilage degradation in osteoarthritis (OA). Treatment of chondrocytes with fibronectin fragments (FN-f) has been used to model OA in vitro, but the system has not been fully characterized. This study sought to define the transcriptional response of chondrocytes to FN-f, and directly compare it to responses traditionally observed in OA. DESIGN: Normal human femoral chondrocytes isolated from tissue donors were treated with either FN-f or PBS (control) for 3, 6, or 18 h. RNA-seq libraries were compared between time-matched FN-f and control samples in order to identify changes in gene expression over time. Differentially expressed genes were compared to a published OA gene set and used for pathway, transcription factor motif, and kinome analysis. RESULTS: FN-f treatment resulted in 3,914 differentially expressed genes over the time course. Genes that are up- or downregulated in OA were significantly up- (P < 0.00001) or downregulated (P < 0.0004) in response to FN-f. Early response genes were involved in proinflammatory pathways, whereas many late response genes were involved in ferroptosis. The promoters of upregulated genes were enriched for NF-κB, AP-1, and IRF motifs. Highly upregulated kinases included CAMK1G, IRAK2, and the uncharacterized kinase DYRK3, while growth factor receptors TGFBR2 and FGFR2 were downregulated. CONCLUSIONS: FN-f treatment of normal human articular chondrocytes recapitulated many key aspects of the OA chondrocyte phenotype. This in vitro model is promising for future OA studies, especially considering its compatibility with genomics and genome-editing techniques.

MiR-146a promotes oligodendrocyte progenitor cell differentiation and enhances remyelination in a model of experimental autoimmune encephalomyelitis.

The death of mature oligodendrocytes (OLs) leads to demyelination in the central nervous system (CNS) and subsequently to functional deficits. Remyelination requires the differentiation of oligodendrocyte progenitor cells (OPCs) into myelinating OLs, which in the CNS with neurodegenerative diseases such as multiple sclerosis (MS), is often inhibited. Among the inhibitors of OPC differentiation are toll-like receptor 2 (TLR2) and interleukin-1 receptor-associated kinase 1 (IRAK1) signaling, and both are negatively regulated by microRNA-146a (miR-146a). Therefore, we hypothesized that increase of miR-146a level in the CNS would foster OPC differentiation and remyelination by inhibiting the TLR2/IRAK1 signaling pathway. Here, we tested this hypothesis using exogenous miR-146a mimics and a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin proteolipid protein peptide (PLP139-151). EAE mice were treated by miR-146a mimics or miR-146a mimic negative controls, respectively, which initiated at day 14 post immunization, once a week for 6 consecutive weeks. Neurological function was monitored daily. Immunofluorescent staining, qRT-PCR and Western blot were used to measure the differentiation of OPCs and myelination, and to investigate the underlying mechanisms of action of miR-146a. Using a fluorescence tracing approach, we found that miR-146a mimics crossed the blood brain barrier and targeted OPCs and microglia/macrophages after systemic administration. MiR-146a mimic treatment substantially improved neurological functional outcome, increased the number of newly generated OLs which may facilitate remyelination in the CNS. The cell number, cytokine level and protein levels of M2 phonotype of microglia/macrophages significantly increased, while cytokine and protein levels of the M1 phenotype significantly decreased after miR-146a mimic treatment. Increased OPC differentiation and remyelination were associated with reduction of TLR2/IRAK1 signaling pathway activity by miR-146a mimic treatment. This study provides insight into the cellular and molecular bases for the therapeutic effects of miR-146a on OPC differentiation and remyelination, and suggests the potential of enhancing miR-146a as a treatment of demyelinating disorders.

Association of microRNA 146 with middle ear hyperplasia in pediatric otitis media.

OBJECTIVE: Toll-like receptor signaling activated by bacterial otitis media pathogens in the middle ear has been shown to play a key role in OM susceptibility, pathogenesis and recovery. Recent studies implicate microRNA 146 (miR-146) in regulation of inflammation via negative feedback of toll-like receptor signaling (TLR) in a wide variety of tissues, however its involvement in otitis media is unknown. METHODS: Human middle ear epithelial cells were stimulated with proinflammatory cytokines, interleukin 1 beta or tumor necrosis factor alpha, for two to twenty-four hours. Middle ear biopsies were collected from children with otitis media with effusion (n = 20), recurrent otitis media (n = 9), and control subjects undergoing cochlear implantation (n = 10). miR-146a, miR-146b expression was assayed by quantitative PCR (qPCR). Expression of miR-146 targets involved in TLR signaling, IRAK1 and TRAF6, was assayed by qPCR in middle ear biopsies. Middle ear biopsies were cryosectioned and epithelial thickness measured by a certified pathologist. RESULTS: Proinflammatory cytokines induced expression of miR-146 in middle ear epithelial cells in vitro. Middle ear miR-146a and miR-146b expression was elevated in otitis media patients relative to control subjects and correlated with middle ear epithelial thickness. A trend towards inverse correlation was observed between miR-146 and TRAF6 expression in the clinical population. CONCLUSIONS: This report is the first to assess miRNA expression in a clinical population with OM. Findings herein suggest miR-146 may play a role in OM.

Pin1-Targeted Therapy for Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease affecting multiple organs in the body, but therapeutic options are still very limited and often come with adverse effects. Increasing evidence has underlined an important role of the Toll-like receptor 7 (TLR-7)/TLR-9/interleukin-1 receptor-associated kinase 1 (IRAK-1)/interferon regulatory factor 7 (IRF-7) pathway in the development and progression of SLE. Notably, the prolyl isomerase Pin1 is an essential regulator of IRAK-1 in TLR-7/TLR-9 signaling, but its role in SLE is unknown. We undertook this study to determine whether Pin1 is activated and plays any role in the development and treatment of SLE. METHODS: Activation of Pin1 and TLR-7/TLR-9/IRAK-1/IRF-7 signaling was determined in various cell types among peripheral blood mononuclear cells from healthy controls and SLE patients. The effects of Pin1 and TLR signaling on SLE development were determined using validated Pin1 short hairpin RNA (shRNA), Pin1 genetic knockout, and the small-molecule Pin1 inhibitor all-trans-retinoic acid (ATRA) in immune cells and in several strains of lupus-prone mice. RESULTS: We found abnormal activation of Pin1 and its downstream targets IRAK-1 and IRF-7 in SLE patients. Furthermore, inhibition of Pin1 using either validated Pin1 shRNA or ATRA blocked TLR-7-induced activation of IRAK-1 and IRF-7 in SLE patient-derived immune cells. Moreover, in multiple lupus-prone animals, both Pin1 knockout and ATRA strikingly attenuated the expression of autoimmunity, including skin lesions, lymphadenopathy, splenomegaly, glomerulonephritis, proteinuria, and production of anti-double-stranded DNA antibodies and CD4-CD8- T cells, and also prolonged overall survival in MRL/lpr and B6.lpr mice. CONCLUSION: Pin1 plays a critical role in the development of SLE, and Pin1-targeted therapy offers a promising new strategy for treating SLE.

Regulation of Transforming Growth Factor ß-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

OBJECTIVE: Transforming growth factor ß-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). METHODS: The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1ß-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA). RESULTS: Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1ß-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination. CONCLUSION: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Histone deacetylase inhibitors increase microRNA-146a expression and enhance negative regulation of interleukin-1ß signaling in osteoarthritis fibroblast-like synoviocytes.

OBJECTIVE: MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1ß (IL-1ß)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). METHOD: MiRNA expression was analyzed using real-time PCR. IL-1ß-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. RESULTS: IL-1ß treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1ß-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. CONCLUSIONS: Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1ß-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.

Lenalidomide efficacy in activated B-cell-like subtype diffuse large B-cell lymphoma is dependent upon IRF4 and cereblon expression.

Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell-like subtype. Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro and delay tumour growth in a human tumour xenograft model, with minimal effect on non-ABC-DLBCL cells. This tumouricidal effect was associated with downregulation of interferon regulatory factor 4 (IRF4), a hallmark of ABC-DLBCL cells. IRF4 inhibition by lenalidomide induced downregulation of B-cell receptor (BCR)-dependent NF-κB. Whereas IRF4-specific small, interfering RNA mimicked the effects of lenalidomide reducing NF-κB activation, IRF4 overexpression enhanced NF-κB activation and conferred resistance to lenalidomide. These findings indicate the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC cells. Furthermore, lenalidomide-induced IRF4 downregulation required the expression of cereblon, a molecular target of lenalidomide. Taken together, these findings suggest that lenalidomide has direct antitumour activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signalling pathway in a cereblon-dependent manner.

miR-146a regulates mechanotransduction and pressure-induced inflammation in small airway epithelium.

Mechanical ventilation generates biophysical forces, including high transmural pressures, which exacerbate lung inflammation. This study sought to determine whether microRNAs (miRNAs) respond to this mechanical force and play a role in regulating mechanically induced inflammation. Primary human small airway epithelial cells (HSAEpCs) were exposed to 12 h of oscillatory pressure and/or the proinflammatory cytokine TNF-α. Experiments were also conducted after manipulating miRNA expression and silencing the transcription factor NF-κB or toll-like receptor proteins IRAK1 and TRAF6. NF-κB activation, IL-6/IL-8/IL-1ß cytokine secretion, miRNA expression, and IRAK1/TRAF6 protein levels were monitored. A total of 12 h of oscillatory pressure and TNF-α resulted in a 5- to 7-fold increase in IL-6/IL-8 cytokine secretion, and oscillatory pressure also resulted in a time-dependent increase in IL-6/IL-8/IL-1ß cytokine secretion. Pressure and TNF-α also resulted in distinct patterns of miRNA expression, with miR-146a being the most deregulated miRNA. Manipulating miR-146a expression altered pressure-induced cytokine secretion. Silencing of IRAK1 or TRAF6, confirmed targets of miR-146a, resulted in a 3-fold decrease in pressure-induced cytokine secretion. Cotransfection experiments demonstrate that miR-146a's regulation of pressure-induced cytokine secretion depends on its targeting of both IRAK1 and TRAF6. MiR-146a is a mechanosensitive miRNA that is rapidly up-regulated by oscillatory pressure and plays an important role in regulating mechanically induced inflammation in lung epithelia.

Kalopanaxsaponin A ameliorates experimental colitis in mice by inhibiting IRAK-1 activation in the NF-κB and MAPK pathways.

BACKGROUND AND PURPOSE: Kalopanaxsaponin A, a triterpenoid saponin isolated from Kalopanax pictus (family Araliaceae), potently inhibited nuclear factor-kappa B (NF-κB) activation in lipopolysaccharide (LPS)-stimulated peritoneal macrophages during a screening programme for anti-colitis agents from natural products. Its anti-inflammatory mechanism remains unknown. Therefore, we investigated its anti-inflammatory effects in lipopolysaccharide (LPS)- or peptidoglycan-stimulated murine peritoneal macrophages and trinitrobenzene sulphonic acid (TNBS)-induced colitic mice. EXPERIMENTAL APPROACH: Peritoneal macrophages from male ICR mice were stimulated with LPS or peptidoglycan in vitro and treated with kalopanaxsaponin A. Colitis was induced in vivo by intrarectal administration of TNBS in male ICR mice. Mice were treated daily with kalopanaxsaponin A, sulphasalazine or phosphate-buffered saline. Inflammatory markers, cytokines, enzymes and transcription factors were measured by ELISA, immunoblot, flow cytometry and immunofluorescent confocal microscopy. KEY RESULTS: Kalopanaxsaponin A potently inhibited the expression of the pro-inflammatory cytokines, interleukin (IL)-1ß, tumour necrosis factor (TNF)-α and IL-6, induced by LPS, but not that induced by TNF-α, in peritoneal macrophages. However, it potently increased the expression of the anti-inflammatory cytokine IL-10. Kalopanaxsaponin A inhibited activation of the IL-1 receptor-associated kinase (IRAK)-1, inhibitor of κB kinase-ß, NF-κB and mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun NH(2) -terminal kinase, p-38), but LPS/Toll-like receptor-4 interaction and IRAK-4 activation were not affected. Oral administration of kalopanaxsaponin A (10 and 20 mg·kg(-1) ) improved the clinical parameters and histology in vivo. Kalopanaxsaponin A inhibited NF-κB and mitogen-activated protein kinase activation induced by TNBS by suppressing IRAK-1 activation. CONCLUSIONS AND IMPLICATIONS: Kalopanaxsaponin A may improve inflammatory diseases, such as colitis, by inhibiting IRAK-1 activation.

Apolipoprotein E interrupts interleukin-1beta signaling in vascular smooth muscle cells.

OBJECTIVES: Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1beta (IL-1beta), a proinflammatory cytokine present in atherosclerotic lesions. METHODS AND RESULTS: IL-1beta-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-kappaB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1beta signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1beta signaling intermediates revealed that NF-kappaB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1beta signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1beta signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-alpha or polyinosinic:polycytidylic acid. CONCLUSION: ApoE targets IRAK-1 activation and thereby interrupts IL-1beta and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Ozonized low density lipoprotein (ozLDL) inhibits NF-kappaB and IRAK-1-associated signaling.

OBJECTIVE: Recent studies have provided strong evidence for the presence of ozone in atherosclerotic lesions. In addition, modification of LDL has been suggested to be involved in atherosclerosis. In the present study we wanted to investigate whether LDL exposed to ozone (ozLDL) is able to modulate the NF-kappaB system, as a paradigm for inflammatory signaling. METHODS AND RESULTS: We showed that activation of NF-kappaB by lipopolysaccharide (LPS), a prototypic inducer of innate immunity, was reversibly inhibited by ozLDL in monocytic THP-1 cells in a dose-dependent manner, whereas tumor necrosis factor (TNF) signaling was not affected. This was not attributable to a direct ozone effect or solely the presence of lipoprotein, and neither required direct contact to LPS nor was accompanied by a change in LPS binding. Comparable inhibitory effects of ozLDL were observed in human monocyte/macrophages and endothelial cells. The presence of ozLDL led to a decrease in LPS-induced IkappaB alpha proteolysis and a reduction of kappaB-dependent transcription/target-gene expression. Furthermore, ozLDL markedly lowered stimulus-induced IkappaB kinase (IKK) activity and phosphorylation/proteolysis of interleukin (IL)-1 receptor-associated kinase-1 (IRAK-1). Finally, cholesterol ozonization products were identified as effective ozLDL inhibitory compounds. CONCLUSIONS: Our study demonstrated that ozLDL inhibited NF-kappaB and IRAK-1-associated signaling which may impair immune function and promote apoptosis.