Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.     

Soluble uric acid induces myocardial damage through activating the NLRP3 inflammasome

Uric acid crystal is known to activate the NLRP3 inflammasome and to cause tissue damages, which can result in many diseases, such as gout, chronic renal injury and myocardial damage. Meanwhile, soluble uric acid (sUA), before forming crystals, is also related to these diseases. This study was carried out to investigate whether sUA could also activate NLRP3 inflammasome in cardiomyocytes and to analyse the mechanisms. The cardiomyocyte activity was monitored, along with the levels of mature IL‐1β and caspase‐1 from H9c2 cells following sUA stimulus. We found that sUA was able to activate NLRP3 inflammasome, which was responsible for H9c2 cell apoptosis induced by sUA. By elevating TLR6 levels and then activating NF‐κB/p65 signal pathway, sUA promoted NLRP3, pro‐caspase 1 and pro‐IL‐1β production and provided the first signal of NLRP3 inflammasome activation. Meanwhile, ROS production regulated by UCP2 levels also contributed to NLRP3 inflammasome assembly and subsequent caspase 1 activation and mature IL‐1β secretion. In addition, the tlr6 knockdown rats suffering from hyperuricemia showed the lower level of IL‐1β and an ameliorative cardiac function. These findings suggest that sUA activates NLRP3 inflammasome in cardiomyocytes and they may provide one therapeutic strategy for myocardial damage induced by sUA.     

Neutralization of pro‐inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C⁺ monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C^hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.     

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.     

Interleukin‐8 and CXCL10 expression in oral keratinocytes and fibroblasts via Toll‐like receptors

Oral keratinocytes and fibroblasts may be the first line of host defense against oral microorganisms. Here, the contention that oral keratinocytes and fibroblasts recognize microbial components via Toll‐like receptors (TLRs) and participate in development of oral inflammation was examined. It was found that immortalized oral keratinocytes (RT7), fibroblasts (GT1) and primary cells express mRNA of TLRs 1–10. Interleukin‐8 (IL‐8) production by RT7 cells was induced by treatment with TLRs 1–9 with the exception of TLR7 agonist, whereas GT1 cells were induced to produce IL‐8 by all TLR agonists tested except for TLR7 and TLR9. GT1 cells showed increased CXCL10 production following treatment with agonists for TLR1/2, TLR3, TLR4, and TLR5, whereas only those for TLR3 and TLR5 increased CXCL10 production in RT7 cells. Moreover, TLR agonists differentially regulated tumor necrosis factor‐alpha‐induced IL‐8 and CXCL10 production by the tested cell types. These findings suggest that recognition of pathogenic microorganisms in oral keratinocytes and fibroblasts by TLRs may have important roles in orchestrating host immune responses via production of various chemokines.     

P2X7 receptor regulation of non‐classical secretion from immune effector cells

P2X7 receptors (P2X7R) are extracellular ATP‐gated ion channels expressed in the immune effector cells that carry out critical protective responses during the early phases of microbial infection or acute tissue trauma. P2X7R‐positive cells include monocytes, macrophages, dendritic cells and T cells. Given its presence in all host and pathogen cell types, ATP can be readily released into extracellular compartments at local sites of tissue damage and microbial invasion. Thus, extracellular ATP and its target receptors on host effector cells can be considered as additional elements of the innate immune system. In this regard, stimulation of P2X7R rapidly triggers a key step of the inflammatory response: induction of NLRP3/caspase‐1 inflammasome signalling complexes that drive the proteolytic maturation and secretion of the proinflammatory cytokines interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18). IL‐1β (and IL‐18) lacks a signal sequence for compartmentation within the Golgi and classical secretory vesicles and the proIL‐1β precursor accumulates within the cytosol following translation on free ribosomes. Thus, ATP‐induced accumulation of the mature IL‐1β cytokine within extracellular compartments requires non‐classical mechanisms of export from the cytosolic compartment. Five proposed mechanisms include: (i) exocytosis of secretory lysosomes that accumulate cytosolic IL‐1β via undefined protein transporters; (ii) release of membrane‐delimited microvesicles derived from plasma membrane blebs formed by evaginationsof the surface membrane that entrap cytosolic IL‐β; (iii) release of membrane‐delimited exosomes secondary to the exocytosis of multivesicular bodies formed by invaginations of recycling endosomes that entrap cytosolic IL‐β; (iv) exocytosis of autophagosomes or autophagolysosomes that accumulate cytosolic IL‐1β via entrapment during formation of the initial autophagic isolation membrane or omegasome and (v) direct release of cytosolic IL‐1β secondary to regulated cell death by pyroptosis or necroptosis. These mechanisms are not mutually exclusive and may represent engagement of parallel or intersecting membrane trafficking responses to P2X7R activation.     

Dexmedetomidine protects against high mobility group box 1‐induced cellular injury by inhibiting pyroptosis

Dexmedetomidine (DEX) is a widely used clinical anesthetic with proven anti‐inflammatory effects. Both high mobility group box 1 (HMGB1) and pyroptosis play an important role in the inflammatory response to infection and trauma. Thus far, there have been no studies published addressing the effect of DEX on HMGB1 and pyroptosis. In order to fill this gap in the literature, bone marrow‐derived macrophages (BMDMs) were exposed to HMGB1 (4 µg/mL) with or without DEX (50 μM) pretreatment. The production of pro‐inflammatory cytokines [such as tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), and IL‐18], phosphorylation of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) and P38, and the activation of caspase‐1 were measured by enzyme immunosorbent assay, western blot analysis, confocal microscope, and flow cytometry, respectively. We found that DEX protected against HMGB1‐induced cell death of BMDMs. In addition, DEX suppressed the generation of TNF‐α, IL‐1β, and IL‐18 as well as the phosphorylation of ERK1/2 and P38. Moreover, DEX inhibited caspase‐1 activation and decreased pyroptosis. Taken together, these findings demonstrate the protective effect of DEX in mediating HMGB1‐induced cellular injury, thus indicating that DEX may be a potential therapeutic candidate for the management of infection and trauma‐derived inflammation.     

P2X7 receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the NLRP3/IL‐1β pathway

Mounting evidence supports the hypothesis that inflammation modulates sympathetic sprouting after myocardial infarction (MI). The myeloid P2X₇ signal has been shown to activate the nucleotide‐binding and oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome, a master regulator of inflammation. We investigated whether P2X₇ signal participated in the pathogenesis of sympathetic reinnervation after MI, and whether NLRP3/interleukin‐1β (IL‐1β) axis is involved in the process. We explored the relationship between P2X₇ receptor (P2X₇R) and IL‐1β in the heart tissue of lipopolysaccharide (LPS)‐primed naive rats. 3′‐O‐(4‐benzoyl) benzoyl adenosine 5′‐triphosphate (BzATP), a P2X₇R agonist, induced caspase‐1 activation and mature IL‐1β release, which was further neutralized by a NLRP3 inhibitor (16673‐34‐0). MI was induced by coronary artery ligation. Following infarction, a marked increase in P2X₇R was localized within infiltrated macrophages and observed in parallel with an up‐regulation of NLRP3 inflammasome levels and the release of IL‐1β in the left ventricle. The administration of A‐740003 (a P2X₇R antagonist) significantly prevented the NLRP3/IL‐1β increase. A‐740003 and/or Anakinra (an IL‐1 receptor antagonist) significantly reduced macrophage infiltration as well as macrophage‐based IL‐1β and NGF (nerve growth factor) production and eventually blunted sympathetic hyperinnervation, as assessed by the immunofluorescence of tyrosine hydroxylase (TH) and growth‐associated protein 43 (GAP 43). Moreover, the use of Anakinra partly attenuated sympathetic sprouting. This indicated that the effect of P2X₇ on neural remodelling was mediated at least partially by IL‐1β. The arrhythmia score of programmed electric stimulation was in accordance with the degree of sympathetic hyperinnervation. In vitro studies showed that BzATP up‐regulated secretion of nerve growth factor (NGF) in M1 macrophages via IL‐1β. Together, these data indicate that P2X₇R contributes to neural and cardiac remodelling, at least partly mediated by NLRP3/IL‐1β axis. Therapeutic interventions targeting P2X₇ signal may be a novel approach to ameliorate arrhythmia following MI.     

IL‐17A promotes cell migration and invasion of glioblastoma cells via activation of PI3K/AKT signalling pathway

Glioblastomas (GBMs) are the most common of both benign and malignant primary brain tumours, in which the inflammatory and immunologic abnormalities are involved. Interleukin‐17A (IL‐17A) plays an important role in various inflammatory diseases and cancers. Several recent studies revealed that the expression of IL‐17A was overexpressed in human GBMs tissue. However, the accurate role of IL‐17A in GBMs remains unclear. In this study, we aimed to explore the effect of IL‐17A on cell migration and invasion of GBMs and the mechanism by which the effects occurred. We found that exogenous IL‐17A promoted significantly cell migration and invasion abilities in two GBMs cell lines (U87MG and U251) in a time‐dependent manner. In addition, the protein expressions of PI3K, Akt and MMP‐2/9 were increased in the GBMs cells challenged by IL‐17A. Furthermore, a tight junction protein ZO‐1 was down‐regulated but Twist and Bmi1 were up‐regulated. Treatment with a PI3K inhibitor (LY294002) significantly reduced the abilities of both migration and invasion in U87MG and U251 cells. LY294002 treatment also attenuated the IL‐17A causing increases of protein levels of PI3K, AKT, MMP‐2/9, Twist and the decreases of protein level of ZO‐1 in the U87MG and U251 cells. Taken together, we concluded that IL‐17A promotes the GBM cells migration and invasion via PI3K/AKT signalling pathway. IL‐17A and its related signalling pathways may be potential therapeutic targets for GBM.     

IL‐1β and TNF‐α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), two pro‐inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL‐1β and/or TNF‐α treatment. Pre‐treatment with N‐Methyl‐d ‐aspartate (NMDA) receptor antagonist MK‐801 blocked cytokine‐induced glutamate production and alleviated the neurotoxicity, indicating that IL‐1β and/or TNF‐α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL‐1β or TNF‐α significantly upregulated the kidney‐type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up‐regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV‐1 encephalitis. In addition, IL‐1β or TNF‐α treatment increased the levels of KGA in cytosol and TNF‐α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.     

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL‐6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well‐studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll‐like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down‐regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up‐regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS‐treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin‐6 (IL‐6), a potent stimulator of cell migration. Interestingly, the levels of IL‐6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental‐derived progenitor cells in sites of periodontal infection.     

IL‐1β Promotes Malignant Transformation and Tumor Aggressiveness in Oral Cancer

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β can be induced by tobacco and betel quid‐related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. 230: 875–884, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Blockade of the PI‐3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro‐inflammatory cytokines

The Aggregatibactor actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol‐3,4,5‐triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP‐1‐ and monocyte‐derived macrophages were found not to be susceptible to Cdt‐induced apoptosis. Nonetheless, the toxin was capable of binding to macrophages and perturbing PI‐3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro‐inflammatory cytokine production including increased expression and release of IL‐1β, TNFα and IL‐6. Furthermore, treatment of cells with either TLR‐2, ‐3 or ‐4 agonists in the presence of Cdt resulted in an augmented pro‐inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt‐induced pro‐inflammatory cytokine response suggesting a pivotal role for PI‐3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt‐producing organisms.     

Overexpression of microRNA‐138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.