The functions of autophagy at the tumour-immune interface.

Autophagy is frequently induced in the hypoxic tumour microenvironment. Accumulating evidence reveals important functions of autophagy at the tumour-immune interface. Herein, we propose an update on the roles of autophagy in modulating tumour immunity. Autophagy promotes adaptive resistance of established tumours to the cytotoxic effects of natural killer cells (NKs), macrophages and effector T cells. Increased autophagic flux in tumours dampen their immunogenicity and inhibits the expansion of cytotoxic T lymphocytes (CTLs) by suppressing the activation of STING type I interferon signalling (IFN-I) innate immune sensing pathway. Autophagy in suppressive tumour-infiltrating immune subsets maintains their survival through metabolic remodelling. On the other hand, autophagy is involved in the antigen processing and presentation process, which is essential for anti-tumour immune responses. Genetic deletion of autophagy induces spontaneous tumours in some models. Thus, the role of autophagy is context-dependent. In summary, our review has revealed the dichotomous roles of autophagy in modulating tumour immunity. Broad targeting of autophagy may not yield maximal benefits. The characterization of specific genes regulating tumour immunogenicity and innovation in targeted delivery of autophagy inhibitors into certain tumours are among the most urgent tasks to sensitize cold cancers to immunotherapy.

miR-506-3p alleviates uncontrolled osteoclastogenesis via repression of RANKL/NFATc1 signaling pathway.

Bone erosion is the major cause of deformities in autoimmune disease conditions such as osteoporosis and rheumatoid arthritis. Aberrant receptor activator of nuclear factor kappa B ligand (RANKL) secretion in bone disorders have been implicated to promote uncontrolled osteoclast differentiation through the regulation of nuclear factor of activated T cells 1 (NFATc1) transcription factor. This phenomenon is governed by several molecular factors including microRNAs, which are under-expressed during disease progression. This report focuses on elucidating the molecular mechanism of miR-506-3p towards the RANKL/NFATc1 pathway. miR-506-3p showed high binding affinity towards NFATc1 (ΔG = -22.4 kcal/mol). Bone marrow-derived macrophages (BMMs) isolated from rats stimulated with RANKL (100 ng/ml) showed active expression of NFATc1 which differentiated into mature osteoclasts. Moreover, NFATc1 activation resulted in downstream secretion of various bone resorptive enzymes (cathepsin K, carbonic anhydrase II, tartarate acid phosphatase, and matrix metalloproteinase 9) which lead to active bone resorption. However, transfection of miR-506-3p resulted in selective repression of NFATc1 inside the cells. This further resulted in the diminished release of bone resorptive enzymes that were essential for the degradation of the bone. Overall, we predict that miR-506-3p can be used as a molecular intervention for RANKL/NFATc1 mediated osteoclastogenesis.

Berberine mitigates IL-21/IL-21R mediated autophagic influx in fibroblast-like synoviocytes and regulates Th17/Treg imbalance in rheumatoid arthritis.

In our previous study, we explored the therapeutic effect of berberine (BBR) against IL-21/IL-21R mediated inflammatory proliferation of adjuvant-induced arthritic fibroblast-like synoviocytes (AA-FLS) through the PI3K/Akt pathway. The current study was designed to explore the therapeutic potential of BBR (15-45 µM) against IL-21/IL-21R mediated autophagy in AA-FLS mediated through PI3K/Akt signaling and Th17/Treg imbalance. Upon IL-21 stimulation, AA-FLS expressed elevated levels of autophagy-related 5 (Atg5), Beclin-1 and LC3-phosphatidylethanolamine conjugate 3-II (LC3-II) through the utilization of p62 and inhibition of C/EBP homologous protein (CHOP). BBR (15-45 µM) inhibited autophagy in AA-FLS cells mediated through PI3K/Akt signaling via suppressing autophagic elements, p62 sequestration and induction of CHOP in a dose-dependent manner. Moreover, IL-21 promoted the uncontrolled proliferation of AA-FLS through induction of B cell lymphoma-2 (Bcl-2) and diminished expression of Bcl-2 associated X protein (BAX) via PI3K/Akt signaling. BBR inhibited the proliferation of AA-FLS via promoting apoptosis through increased expression of BAX and diminished Bcl-2 transcription factor levels. Furthermore, T cells stimulated with IL-21 induced CD4+ CD196+ Th17 cells proliferation through RORγt activation mediated in a PI3K/Akt dependent manner. BBR inhibited the proliferation of Th17 cells through downregulation of RORγt in a concentration-dependent manner. BBR also promoted the differentiation of CD4+ CD25+ Treg cells through induction of forkhead box P3 (Foxp3) activation via aryl hydrocarbon receptor (AhR) and upregulation of cytochrome P450 family 1, subfamily A, polypeptide 1 (CYP1A1). Collectively, we conclude that BBR might attenuate AA-FLS proliferation through inhibition of IL-21/IL-21R dependent autophagy and regulates the Th17/Treg imbalance in RA.

Multifaceted role of IL-21 in rheumatoid arthritis: Current understanding and future perspectives.

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disorder designated with hyperplastic synovium, bone destruction and cartilage degradation. Current therapies involve targeting major cytokines and inflammatory mediators involved in RA to alleviate the pain and provide a temporary relief. Interleukin 21 (IL-21), a recently identified cytokine is known to possess a versatile role in modulating the cells of the RA synovium. Over the past decade, the pleiotropic role of IL-21 in RA pathogenesis has been implicated in several aspects. T helper 17 (Th17) and follicular T helper cells (Tfh), being the key immunomodulators of the RA synovium secrete high amounts of IL-21 during disease progression. Several studies have provided experimental evidences elucidating the multifaceted role of IL-21 in RA disease progression. IL-21 has the potential to activate T cells, B cells, monocytes/macrophages and synovial fibroblasts in RA pathogenesis through activation of JAK-STAT, MAPK and PI3K/Akt signaling pathways. Till date, therapies targeting Th17 cells and its inflammatory cytokines have been under investigation and are subjected to various clinical trials. This review showcases the role of IL-21 in RA pathogenesis and recent reports implicating its function in various immune cells, major signaling pathways, and in promoting osteoclastogenesis.

Berberine inhibits IL-21/IL-21R mediated inflammatory proliferation of fibroblast-like synoviocytes through the attenuation of PI3K/Akt signaling pathway and ameliorates IL-21 mediated osteoclastogenesis.

The current study investigated the therapeutic effect of berberine (BBR), an alkaloid derivative against IL-21/IL-21R mediated phosphotidyl inositol 3 kinase/protein kinase B (PI3K/Akt) signaling in adjuvant induced arthritic fibroblast-like synoviocytes (AA-FLS) isolated from rats and IL-21 mediated osteoclastogenesis in bone-marrow derived monocytes (BMMs). BBR (15-45 µM) treatment attenuated the gene and protein levels of IL-21R complex. BBR suppressed the levels of IL-21 (20 ng/ml) mediated production of inflammatory cytokines such as: tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6) and interleukin 23 (IL-23) in AA-FLS cells. Subsequently, BBR ameliorated the gene and protein expression levels of mechanistic target of rapamycin (mTOR), IL-23 and nuclear factor kappa B (NFκB) p65 through the inhibition of PI3K and upregulation of phosphatase and tensin homolog (PTEN) at the protein level. Furthermore, BBR also inhibited the phosphorylation of Akt and NFκB p65 in a dose dependant manner. LY294002 (20 µM) treatment suppressed the PI3K/Akt signaling and its downstream elements in AA-FLS cells. BBR also modulated IL-21 mediated osteoclastogenesis through the suppression of PI3K dependant nuclear factor of activated T-cells 1 (Nfatc1) induction. Moreover, BBR controlled the osteoclast differentiation via inhibition of various bone resorptive enzymes including: cathepsin K, matrix metalloproteinase 9 (MMP9) and tartarate acid phosphatase (TRAP). LY294002 also inhibited osteoclast formation via suppression of PI3K mediated Nfatc1 induction and other downstream elements. Overall, our findings suggest that BBR is a potential candidate for therapeutic targeting of IL-21/IL-21R mediated RA pathogenesis.

Berberine modulates ASK1 signaling mediated through TLR4/TRAF2 via upregulation of miR-23a.

The current study was designed to explore the underlying therapeutic effect of berberine (BBR), an alkaloid compound against LPS (1 µg/ml)/TNFα (10 ng/ml) mediated apoptosis signal-regulating kinase 1 (ASK1) signaling in RAW 264.7 macrophages and adjuvant-induced arthritic synovial macrophages (AA-SM) with relation to miR-23a levels. LPS and TNFα stimulation abrogated the expression of miR-23a resulting in TLR4/TRAF2 mediated ASK1 activation and downstream phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). BBR (25-75 µM) treatment ameliorated the gene expression levels of TLR4, TRAF2, TNFα, IL-6, and IL-23 through the upregulation of miR-23a. Subsequently, BBR suppressed the levels of TLR4/TRAF2 mediated phosphorylation of ASK1/p38 and attenuated the expression of various pro-inflammatory cytokines (TNFα, IL-6 & IL-23) in RAW 264.7 macrophages and AA-SM cells. BBR was able to counteract these factors through activation of miR-23a levels in LPS/TNFα stimulated RAW 264.7 macrophages and AA-SM cells. NQDI1 (30 µM) treatment inhibited ASK1 activation resulting in basal levels of miR-23a, owing to the conclusion that ASK1 activation downregulates miR-23a levels inside the cells. Overall, our current findings predict that BBR is a potential candidate for therapeutic targeting of TLR4/TRAF2 mediated ASK1 activation in inflammatory and in RA pathogenesis possibly through post-transcriptional gene silencing via upregulation of miR-23a.

uPA/uPAR signaling in rheumatoid arthritis: Shedding light on its mechanism of action.

Rheumatoid arthritis (RA) is a systemic and chronic autoimmune inflammatory disorder affecting multiple joints. Various cytokines, chemokines and growth factors synergistically modulate the joint physiology leading to bone erosion and cartilage degradation. Other than these conventional mediators that are well established in the past, the newly identified plasminogen activator (PA) family of proteins have been witnessed to possess a multifactorial approach in mediating RA pathogenesis. One such family of proteins comprises of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR)/soluble-type plasminogen activator receptor (suPAR). PA family of proteins are classified into two types namely: uPA and tissue type plasminogen activator (tPA). Both these subtypes have been implicated to play a key role in RA disease progression. However during RA pathogenesis, uPA secreted by neutrophils, chondrocytes, and monocytes are designated to interact with uPAR expressed on macrophages, fibroblast-like synoviocytes (FLS), chondrocytes and endothelial cells. Interaction of uPA/uPAR promotes the disease progression of RA through secretion of several cytokines, chemokines, growth factors and matrix metalloproteinases (MMPs). Moreover, uPA/uPAR initiates inflammatory responses in macrophages and FLS through activation of PI3K/Akt signaling pathways. Furthermore, uPAR plays a dual role in osteoclastogenesis under the presence/absence of growth factors like monocyte-colony stimulating factor (M-CSF). Overall, this review emphasizes the role of uPA/uPAR on various immune cells, signaling pathways and osteoclastogenesis involved in RA pathogenesis.

MicroRNA-532-3p Regulates Pro-Inflammatory Human THP-1 Macrophages by Targeting ASK1/p38 MAPK Pathway.

Inflammation is a complex biological process which alters the normal physiological function of the immune system resulting in an abnormal microenvironment that leads to several clinical complications. The process of inflammation is mediated through various intracellular signaling factors inside the cells. Apoptosis signal-regulating kinase 1 (ASK1) is an inflammation-derived kinase that controls the activation of other family of kinases such as p38 mitogen-activated protein kinases (p38 MAPKs), which mediates various the inflammatory processes. In this study, we cultured THP-1 macrophage cells to undergo inflammatory proliferation with LPS (1 µg/ml) and TNFα (10 ng/ml) stimulation. Initial in silico analysis was utilized to predict novel microRNAs (miRNAs) that target ASK1 signaling and its expression levels in LPS and TNFα stimulated THP-1 cells were estimated. Among the miRNAs, miR-532-3p showcased the highest binding affinity towards ASK1 kinase. We witnessed that transient transfection of miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK. Furthermore, direct targeting of ASK1 resulted in regulation of uncontrolled release of cytokines (TNFα, IL-6, and IL-23) and chemokines (GM-CSF and MIP-2α). Overall, we suggest that miR-532-3p attenuates the pro-inflammatory nature of macrophages by targeting ASK1/p38 MAPK signaling pathway and can be used as a molecular intervention for treating inflammatory diseases.

Berberine encapsulated PEG-coated liposomes attenuate Wnt1/ß-catenin signaling in rheumatoid arthritis via miR-23a activation.

Bone erosion is a debilitating pathological process of osteopathic disorder like rheumatoid arthritis (RA). Current treatment strategies render low disease activity but with disease recurrence. To find an alternative, we designed this study with an aim to explore the underlying therapeutic effect of PEGylated liposomal BBR (PEG-BBR) against Wnt1/ß-catenin mediated bone erosion in adjuvant-induced arthritic (AA) rat model and fibroblast-like synoviocytes (FLS) with reference to microRNA-23a (miR-23a) activity. Our initial studies using confocal microscopy and Near-Infrared Imaging (NIR) showed successful internalization of PEG-BBR and PEG-miR-23a in vitro and in vivo respectively and was retained till 48 h. The preferential internalization of PEG-BBR into the inflamed joint region significantly reduced the gene and protein level expression of major Wnt1 signaling mediators and reduced bone erosion in rats. Moreover, PEG-BBR treatment in FLS cells attenuated the gene and protein expression levels of FZD4, LRP5, ß-catenin, and Dvl-1 through the induction of CYLD. Furthermore, inhibition of these factors resulted in reduced bone loss and increased calcium retainability by altering the RANKL/OPG axis. PEG-BBR treatment markedly inhibited the expression of LRP5 protein on par with the DKK-1 (LRP5/Wnt signaling inhibitor) and suppressed the transcriptional activation of ß-catenin inside the cells. We further witnessed that miR-23a altered the expression levels of LRP5 through RNA interference. Overall, our findings endorsed that miR-23a possesses a multifaceted therapeutic efficiency like berberine in RA pathogenesis and can be considered as a potential candidate for therapeutic targeting of Wnt1/ß-catenin signaling in RA disease condition.

MiR-145-5p mitigates dysregulated Wnt1/ß-catenin signaling pathway in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) lining the arthritic synovial joint region have been implicated to be a key player in bone remodeling. The uncontrolled proliferation of this cell subtype is strictly regulated by various molecular elements including microRNAs (miRNAs). The Wnt1/ß-catenin signaling pathway plays a crucial role in the survival of FLS cells. This study explores the underlying mechanism of miR-145-5p towards the Wnt1/ß-catenin pathway. MiR-145-5p depicted a strong binding affinity towards frizzled class receptor 4 (FZD4) 3' UTR, a key receptor complex essential for recognizing circulating Wnt1 molecules. Adjuvant induced arthritic fibroblast-like synoviocytes (AA-FLS) isolated from rats stimulated with Wnt1 (10 ng/ml) elicited active Wnt1/ß-catenin signaling. Transfection of miR-145-5p mimic (50 pmol) to AA-FLS stimulated with Wnt1 elicited reduced expression levels of various factors of Wnt1/ß-catenin signaling including low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled segment polarity protein 1 (Dvl1) and ß-catenin transcription factor. Moreover, pro-inflammatory cytokines (TNFα, IL-1ß, IL-6 and IL-23) were regulated compared to the diseased groups. Furthermore, miR-145-5p counterbalanced the levels of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) at the cellular level, essential for bone remodeling. Hence, we suggest that miR-145-5p regulates the survival/proliferation of FLS cells in RA disease condition through attenuation of Wnt1/ß-catenin signaling.

Berberine, an isoquinoline alkaloid suppresses TXNIP mediated NLRP3 inflammasome activation in MSU crystal stimulated RAW 264.7 macrophages through the upregulation of Nrf2 transcription factor and alleviates MSU crystal induced inflammation in rats.

The current study was designed to investigate the therapeutic potential of berberine on monosodium urate (MSU) crystal stimulated RAW 264.7 macrophages and in MSU crystal induced rats. Our results indicate that berberine (25, 50 and 75µM) suppressed the levels of pro-inflammatory cytokines (interleukin-1beta (IL-1ß) and tumor necrosis factor alpha (TNFα)) and intracellular reactive oxygen species in MSU crystal stimulated RAW 264.7 macrophages. The mRNA expression levels of IL-1ß, caspase 1, nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), thioredoxin interacting protein (TXNIP) and kelch-like ECH-associated protein 1 (Keap1) were found downregulated with the upregulation of nuclear factor erythroid-2-related factor 2 (Nrf2) transcription factor and its associated anti-oxidant enzymes: Heme oxygenase I (HO-1), superoxide dismutase (SOD1), glutathione peroxidase (GPx), NADPH quinone oxidoreductase-1 (NQO1) and catalase (CAT) in MSU crystal stimulated RAW 264.7 macrophages upon berberine treatment. Subsequently, western blot analysis revealed that berberine decreased the protein expression of IL-1ß and caspase 1 and increased Nrf2 expression in RAW 264.7 macrophages. Immunofluorescence analysis also explored increased expression of Nrf2 in MSU crystal stimulated RAW 264.7 macrophages by berberine treatment. In addition, the paw edema, pain score, pro-inflammatory cytokines (IL-1ß and TNFα) and articular elastase activity were found significantly reduced in berberine (50mg/kgb·wt) administered MSU crystal-induced rats. Conclusively, our current findings suggest that berberine may represent as a potential candidate for the treatment of gouty arthritis by suppressing inflammatory mediators and activating Nrf2 anti-oxidant pathway.

Cytotoxicity and immunomodulatory effects of sol-gel combustion based titanium dioxide (TiO2) particles of large surface area on RAW 264.7 macrophages.

The current study was designed to investigate the cytotoxicity and immunomodulatory effects of sol-gel combustion based TiO2 particles (glycine and l-alanine as reducing agents) of large surface area on RAW 264.7 macrophages. RAW 264.7 macrophages exposed to varying concentrations of TiO2 particles (0.001 to 1000µg/ml) were assessed after 24h and showed a reduced cell viability at 100 and 1000µg/ml and increased LDH release at 10µg/ml. Furthermore, TiO2 particles (0.1, 1 and 10µg/ml) were utilized to assess the immune responses and intracellular ROS levels on RAW 264.7 macrophages. TiO2 particles at 10µg/ml showed increased mRNA expression of inflammatory cytokines (TNFα, IL-1ß and IL-6), inflammatory mediators (iNOS and COX-2) and transcription factor (NFκB) similar to that of LPS stimulated macrophages. However, the mRNA expression levels were found near normal levels at lower concentrations (0.1 and 1µg/ml). In addition, TiO2 particles at 10µg/ml also increased the production of inflammatory cytokines (TNFα, IL-1ß and IL-6) and intracellular ROS levels in RAW 264.7 macrophages similar to that of LPS stimulated macrophages. Conclusively, TiO2 particles prepared through this method at a concentration≤0.1µg/ml can be used for various biological applications with minimal immunomodulatory effects.