Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

Intermittent hypoxia regulates stem-like characteristics and differentiation of neuroblastoma cells

Background

Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells.

Methods and Findings

Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells.

Conclusions

Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.     

Why CCR2 and CCR5 blockade failed and why CCR1 blockade might still be effective in the treatment of rheumatoid arthritis

Background

The aim of this study was to provide more insight into the question as to why blockade of CCR1, CCR2, and CCR5 may have failed in clinical trials in rheumatoid arthritis (RA) patients, using an in vitro monocyte migration system model.

Methodology/Principal Findings

Monocytes from healthy donors (HD; n = 8) or from RA patients (for CCR2 and CCR5 antibody n = 8; for CCR1 blockade n = 13) were isolated from peripheral blood and pre-incubated with different concentrations of either anti-CCR1, anti-CCR2, or anti-CCR5 blocking antibodies (or medium or isotype controls). In addition, a small molecule CCR1 antagonist (BX471) was tested. Chemotaxis was induced by CCL2/MCP-1 (CCR2 ligand), CCL5/RANTES (CCR1 and CCR5 ligand), or by a mix of 5 RA synovial fluids (SFs), and cellular responses compared to chemotaxis in the presence of medium alone. Anti-CCR2 antibody treatment blocked CCL2/MCP-1-induced chemotaxis of both HD and RA monocytes compared to isotype control. Similarly, anti-CCR5 antibody treatment blocked CCL5/RANTES-induced chemotaxis of RA monocytes. While neither CCR2 nor CCR5 blocking antibodies were able to inhibit SF-induced monocyte chemotaxis, even when both receptors were blocked simultaneously, both anti-CCR1 antibodies and the CCR1 antagonist were able to inhibit SF-induced monocyte chemotaxis.

Conclusions/Significance

The RA synovial compartment contains several ligands for CCR1, CCR2, and CCR5 as well as other chemokines and receptors involved in monocyte recruitment to the site of inflammation. The results suggest that CCR2 and CCR5 are not critical for the migration of monocytes towards the synovial compartment in RA. In contrast, blockade of CCR1 may be effective. Conceivably, CCR1 blockade failed in clinical trials, not because CCR1 is not a good target, but because very high levels of receptor occupancy at all times may be needed to inhibit monocyte migration in vivo.     

The CCL2/CCR2 Axis Enhances Vascular Cell Adhesion Molecule-1 Expression in Human Synovial Fibroblasts

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of osteoarthritis (OA). Here, we investigated the intracellular signaling pathways involved in CCL2-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human OA synovial fibroblasts (OASFs).

Methodology/Principal Findings

Stimulation of OASFs with CCL2 induced VCAM-1 expression. CCL2-mediated VCAM-1 expression was attenuated by CCR2 inhibitor (RS102895), PKCδ inhibitor (rottlerin), p38MAPK inhibitor (SB203580), and AP-1 inhibitors (curcumin and tanshinone IIA). Stimulation of cells with CCL2 increased PKCδ and p38MAPK activation. Treatment of OASFs with CCL2 also increased the c-Jun phosphorylation and c-Jun binding to the AP-1 element on the VCAM-1 promoter. Moreover, CCL2-mediated CCR2, PKCδ, p38MAPK, and AP-1 pathway promoted the adhesion of monocytes to the OASFs monolayer.

Conclusions/Significance

Our results suggest that CCL2 increases VCAM-1 expression in human OASFs via the CCR2, PKCδ, p38MAPK, c-Jun, and AP-1 signaling pathway. The CCL2-induced VCAM-1 expression promoted monocytes adhesion to human OASFs.     

Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

Background

Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions.

Methods

We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone.

Results

Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells.

Conclusions

Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.     

Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice

Background

Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.

Methodology/Principal Findings

We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C^hi/CCR2^hi monocytes. Surprisingly, neutrophils, not Ly6C^lo monocytes, largely replaced Ly6C^hi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.

Conclusion/Significance

These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.     

The pathological effects of CCR2+ inflammatory monocytes are amplified by an IFNAR1-triggered chemokine feedback loop in highly pathogenic influenza infection

Background

Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection.

Results

We established H1N1 IAV-infected mouse models using three viruses of varying pathogenicity and noted the accumulation of a defined Gr1 + CD11b + myeloid population correlating with the pathogenicity. Herein, we reported that CCR2+ inflammatory monocytes are the major cell compartments in this population. Of note, impaired clearance of the high pathogenicity virus prolonged IFN expression, leading to CCR2+ inflammatory monocytes amplifying their own recruitment via an interferon-α/β receptor 1 (IFNAR1)-triggered chemokine loop. Blockage of IFNAR1-triggered signaling or inhibition of viral replication by Oseltamivir significantly suppresses the expression of CCR2 ligands and reduced the influx of CCR2+ inflammatory monocytes. Furthermore, trafficking of CCR2+ inflammatory monocytes from the bone marrow to the lung was evidenced by a CCR2-dependent chemotaxis. Importantly, leukocyte infiltration, cytokine storm and expression of iNOS were significantly reduced in CCR2−/− mice lacking infiltrating CCR2+ inflammatory monocytes, enhancing the survival of the infected mice.

Conclusions

Our results indicated that uncontrolled viral replication leads to excessive production of inflammatory innate immune responses by accumulating CCR2+ inflammatory monocytes, which contribute to the fatal outcomes of high pathogenicity virus infections.     

An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

Background

Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.

Methodology

The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.

Conclusions/Findings

MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.     

Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity

Background

Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization.

Methodology/Principal Findings

We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the α_2A-adrenoceptor (α_2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14''304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of α_2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation.

Conclusions/Significance

Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.     

Rhodomyrtone Modulates Innate Immune Responses of THP-1 Monocytes to Assist in Clearing Methicillin-Resistant Staphylococcus aureus

Background

The increasing resistance of Staphylococcus aureus to conventional antibiotics poses a major health problem. Moreover, S. aureus can survive within phagocytes, thus evading some antibiotics and the innate immune response. Rhodomyrtone, a bioactive compound from the leaves of Rhodomyrtus tomentosa, possesses potent antibacterial activity against methicillin-resistant S. aureus (MRSA). This study was to investigate the immunomodulatory effects of rhodomyrtone on THP-1 monocytes in response to MRSA.

Methods

THP-1 monocytes were stimulated with heat-killed MRSA, followed by treatment with rhodomyrtone. The cell pellets were prepared to detect pro-inflammatory molecules using real-time PCR. The supernatants were collected to assess nitric oxide production using Griess assay. Assays for phagocytosis and bacterial killing by THP-1 monocytes were performed to determine if they were affected by rhodomyrtone.

Results

Expression of pro-inflammatory molecules including IL-1β, TNF-α, IL-6, and iNOS was enhanced in THP-1 monocytes stimulated with high doses of heat-killed MRSA (10⁸ to 10⁹ cfu/ml). In contrast, monocytes stimulated with MRSA at lower doses (10⁶ to 10⁷ cfu/ml) did not induce the expression of these cytokines. However, rhodomyrtone significantly increased the expression of pro-inflammatory mediators, IL-6 and iNOS in monocytes stimulated with heat-killed MRSA at low doses, and displayed some anti-inflammatory activity by reducing TNF-α expression in monocytes stimulated with heat-killed MRSA at high doses. Treatment with rhodomyrtone also significantly up-regulated the expression of the key pattern recognition receptors, TLR2 and CD14, in THP-1 monocytes stimulated with heat-killed MRSA at 10⁶ to 10⁹ cfu/ml, while heat-killed MRSA alone did not induce the expression of these molecules. The ability of rhodomyrtone to eliminate MRSA from the monocytes was observed within 24 h after treatment.

Conclusion

Rhodomyrtone enhanced the expression of pattern recognition receptors by monocytes in response to MRSA. Increased expression of these receptors might improve MRSA clearance by modulating pro- and anti-inflammatory cytokine responses.     

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion,Transendothelial Migration and Phagocytosis

Background

Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β₂ integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis.

Methodology/Principal Findings

THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β₂ integrin cell surface expression and β₂ integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β₂ integrins, in particular CD11b/CD18. SIRPα overexpression reduced β₂ integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β₂ integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression.

Conclusions/Significance

SIRPα negatively regulates β₂ integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.     

Interleukin-32-induced thymic stromal lymphopoietin plays a critical role in macrophage differentiation through the activation of caspase-1 in vitro

Introduction

Interleukin (IL)-32 is an inflammatory cytokine induced by Mycobacterium tuberculosis and Mycobacterium bovis in a variety of cell types and discovered in the synovial of patients with rheumatoid arthritis (RA). Thymic stromal lymphopoietin (TSLP) play several roles in the pathogenesis of RA. However, the role of IL-32 and TSLP in RA has not been elucidated.

Methods

We evaluated the specific mechanism of between IL-32 and TSLP in RA using human monocyte cell line, THP-1 cells.

Results

Here we documented for the first time that IL-32 highly increased TSLP production in THP-1 cells and human blood monocytes. TSLP expression was induced by IL-32 via activation of caspase-1 and nuclear factor-κB. TSLP produced by IL-32 increased differentiation of monocytes but depletion of TSLP prevented differentiation of monocytes into macrophage-like cells. Chondroprotective drugs such as chondroitin sulfate (CS) and the traditional Korean medicine, BaekJeol-Tang (BT) decrease production of TSLP and activation of caspase-1 and nuclear factor-κB. In addition, CS and BT inhibited IL-32-induced monocytes differentiation.

Conclusions

Taken together, IL-32 and TSLP are important cytokines involved in the development of RA. The effects of CS and BT were associated with the downregulation of TSLP and caspase-1 through negative regulation of IL-32 pathways in RA.     

Interleukin-1 receptor-associated kinase-3 is a key inhibitor of inflammation in obesity and metabolic syndrome

Background

Visceral obesity is associated with the rising incidence of type 2 diabetes and metabolic syndrome. Low-grade chronic inflammation and oxidative stress synergize in obesity and obesity-induced disorders.

Objective

We searched a cluster of molecules that support interactions between these stress conditions in monocytes.

Methods

RNA expressions in blood monocytes of two independent cohorts comprising 21 and 102 obese persons and 46 age-matched controls were determined by microarray and independently validated by quantitative RT-PCR analysis. The effect of three-month weight loss after bariatric surgery was determined. The effect of RNA silencing on inflammation and oxidative stress was studied in human monocytic THP-1 cells.

Results

Interleukin-1 receptor-associated kinase-3 (IRAK3), key inhibitor of IRAK/NFκB-mediated chronic inflammation, is downregulated in monocytes of obese persons. Low IRAK3 was associated with high superoxide dismutase-2 (SOD2), a marker of mitochondrial oxidative stress. A comparable expression profile was also detected in visceral adipose tissue of the same obese subjects. Low IRAK3 and high SOD2 was associated with a high prevalence of metabolic syndrome (odds ratio: 9.3; sensitivity: 91%; specificity: 77%). By comparison, the odds ratio of high-sensitivity C-reactive protein, a widely used marker of systemic inflammation, was 4.3 (sensitivity: 69%; specificity: 66%). Weight loss was associated with an increase in IRAK3 and a decrease in SOD2, in association with a lowering of systemic inflammation and a decreasing number of metabolic syndrome components. We identified the increase in reactive oxygen species in combination with obesity-associated low adiponectin and high glucose and interleukin-6 as cause of the decrease in IRAK3 in THP-1 cells in vitro.

Conclusion

IRAK3 is a key inhibitor of inflammation in association with obesity and metabolic syndrome. Our data warrant further evaluation of IRAK3 as a diagnostic and prognostic marker, and as a target for intervention.     

IFI16 protein mediates the anti-inflammatory actions of the type-I interferons through suppression of activation of caspase-1 by inflammasomes

Background

Type-I interferons (IFNs) are used to treat certain inflammatory diseases. Moreover, activation of type-I IFN-signaling in immune cells inhibits the production of proinflammatory cytokines and activation of inflammasomes. However, the molecular mechanisms remain largely unknown. Upon sensing cytosolic double-stranded DNA, the AIM2 protein forms the AIM2-ASC inflammasome, resulting in activation of caspase-1. Given that the IFI16 and AIM2 proteins are IFN-inducible and can heterodimerize with each other, we investigated the regulation of IFI16, AIM2, and inflammasome proteins by type-I and type-II IFNs and explored whether the IFI16 protein could negatively regulate the activation of the AIM2 (or other) inflammasome.

Methodology/ Principal Findings

We found that basal levels of the IFI16 and AIM2 proteins were relatively low in peripheral blood monocytes (CD14⁺) and in the THP-1 monocytic cell line. However, treatment of THP-1 cells with type-I (IFN-α or β) or type-II (IFN-γ) IFN induced the expression levels of IFI16, AIM2, ASC and CASP1 proteins. The induced levels of IFI16 and AIM2 proteins were detected primarily in the cytoplasm. Accordingly, relatively more IFI16 protein bound with the AIM2 protein in the cytoplasmic fraction. Notably, increased expression of IFI16 protein in transfected HEK-293 cells inhibited activation of caspase-1 by the AIM2-ASC inflammasome. Moreover, the constitutive knockdown of the IFI16 expression in THP-1 cells increased the basal and induced [induced by poly(dA:dT) or alum] activation of the caspase-1 by the AIM2 and NLRP3 inflammasomes.

Conclusions/Significance

Our observations revealed that the type-I and type-II IFNs induce the expression of IFI16, AIM2, and inflammasome proteins to various extents in THP-1 cells and the expression of IFI16 protein in THP-1 cells suppresses the activation of caspase-1 by the AIM2 and NLRP3 inflammasomes. Thus, our observations identify the IFI16 protein as a mediator of the anti-inflammatory actions of the type-I IFNs.     

Weight and metabolic effects of CPAP in obstructive sleep apnea patients with obesity

Background

Obstructive sleep apnea (OSA) is associated with obesity, insulin resistance (IR) and diabetes. Continuous positive airway pressure (CPAP) rapidly mitigates OSA in obese subjects but its metabolic effects are not well-characterized. We postulated that CPAP will decrease IR, ghrelin and resistin and increase adiponectin levels in this setting.

Methods

In a pre- and post-treatment, within-subject design, insulin and appetite-regulating hormones were assayed in 20 obese subjects with OSA before and after 6 months of CPAP use. Primary outcome measures included glucose, insulin, and IR levels. Other measures included ghrelin, leptin, adiponectin and resistin levels. Body weight change were recorded and used to examine the relationship between glucose regulation and appetite-regulating hormones.

Results

CPAP effectively improved hypoxia. However, subjects had increased insulin and IR. Fasting ghrelin decreased significantly while leptin, adiponectin and resistin remained unchanged. Forty percent of patients gained weight significantly. Changes in body weight directly correlated with changes in insulin and IR. Ghrelin changes inversely correlated with changes in IR but did not change as a function of weight.

Conclusions

Weight change rather than elimination of hypoxia modulated alterations in IR in obese patients with OSA during the first six months of CPAP therapy.     

CXCL1 Contributes to β-Amyloid-Induced Transendothelial Migration of Monocytes in Alzheimer’s Disease

Background

Bone marrow-derived microglia that originates in part from hematopoietic cells, and more particularly from monocytes preferentially attach to amyloid deposition in brains of Alzheimer’s disease (AD). However, the mechanism of monocytes recruited into the amyloid plaques with an accelerated process in AD is unclear.

Methodology/Principal Findings

Here we reported that monocytes from AD patients express significantly higher chemokine (C-X-C motif) ligand 1 (CXCL1) compared to age-matched controls. AD patient’s monocytes or CXCL1-overexpressing THP-1 cells had enhanced ability of β-amyloid (Aβ)-induced transendothelial migration and Aβ-induced transendothelial migration for AD patient’s monocytes or CXCL1-overexpressing THP-1 cells was almost abrogated by anti-CXCL1 antibody. Furthermore, monocytes derived from a transgenic mouse model of AD also expressed significantly higher CXCL1. CD11b⁺CD45^hi population of cells that were recruited from the peripheral blood were markedly bolcked in APP mouse brain by anti-CXCL1 antibody. Accordingly, in response to Aβ, human brain microvascular endothelial cells (HBMEC) significantly up-regulated CXC chemokine receptor 2 (CXCR2) expression, which was the only identified receptor for CXCL1. In addition, a high level expression of CXCR2 in HBMEC significantly promoted the CXCL1-overexpressing THP-1 cells transendothelial migration, which could be was abrogated by anti-CXCR2 antibody. Further examination of possible mechanisms found that CXCL1-overexpressing THP-1 cells induced transendothelial electrical resistance decrease, horseradish peroxidase flux increase, ZO-1 discontinuous and occludin re-distribution from insoluble to soluble fraction through interacting with CXCR2. ROCK inhibitor, Y27632, could block CXCL1-overexpressing THP-1 cells transendothelial migration, whereas other inhibitors had no effects.

Conclusions/Significance

The present data indicate that monocytes derived from AD patients overexpressing CXCL1, which is a determinant for Aβ-induced transendothelial migration. CXCL1 expressed by monocytes and CXCR2 on HBMEC is involved in monocytes migrating from blood to brain in AD patients.     

Unlike for human monocytes after LPS activation, release of TNF-α by THP-1 cells is produced by a TACE catalytically different from constitutive TACE

Background

Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α.

Methodology/Principal Findings

Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation.

Conclusions/Significance

On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.     

Decrease of miR-146b-5p in monocytes during obesity is associated with loss of the anti-inflammatory but not insulin signaling action of adiponectin

Background

Low adiponectin, a well-recognized antidiabetic adipokine, has been associated with obesity-related inflammation, oxidative stress and insulin resistance. Globular adiponectin is an important regulator of the interleukin-1 receptor-associated kinase (IRAK)/NFκB pathway in monocytes of obese subjects. It protects against inflammation and oxidative stress by inducing IRAK3. microRNA (miR)-146b-5p inhibits NFκB-mediated inflammation by targeted repression of IRAK1 and TNF receptor-associated factor-6 (TRAF6). Therefore, we measured the expression of miR-146b-5p in monocytes of obese subjects. Because it was low we determined the involvement of this miR in the anti-inflammatory, antioxidative and insulin signaling action of globular adiponectin.

Methods

miR-146b-5p expression in monocytes of obese subjects was determined by qRT-PCR. The effect of miR-146b-5p silencing on molecular markers of inflammation, oxidative stress and insulin signaling and the association with globular adiponectin was assessed in human THP-1 monocytes.

Results

miR-146b-5p was downregulated in monocytes of obese persons. Low globular adiponectin decreased miR-146b-5p and IRAK3 in THP-1 monocytes, associated with increased mitochondrial reactive oxygen species (ROS). Intracellular ROS and insulin receptor substrate-1 (IRS1) protein were unchanged. Silencing of miR-146b-5p with an antisense inhibitor resulted in increased expression of IRAK1 and TRAF6 leading to more NFκB p65 DNA binding activity and TNFα. As a response IRAK3 and IRS1 protein increased. Mitochondrial and intracellular ROS production did not increase despite more inflammation. In addition, exposure of miR-146b-5p-depleted THP-1 monocytes to high levels of globular adiponectin resulted in an increased production of TNFα and intracellular ROS. Still, they did not lose their potential to increase IRAK3 and IRS1 protein and to decrease mitochondrial ROS.

Conclusion

miR-146b-5p, decreased in monocytes during obesity, is a major mediator of the anti-inflammatory action of globular adiponectin. It appears not to be involved in insulin signaling possibly by protective response of IRAK3 and lack of mitochondrial ROS production.     

The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production

Background

The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system.

Principal Findings

NLRP3-WT and NLRP3-Q705K were retrovirally transduced into the human monocytic cell line THP-1, followed by the assessment of IL-1β and IL-18 levels in the cell culture supernatant. THP-1 cells expressing the above NLRP3 variants were sorted based upon Green Fluorescent Protein (GFP) expression. Cytokine response to alum (one of the most widely used adjuvants in vaccines) in the cells stably expressing NLRP3-WT and NLRP3-Q705K were determined. IL-1β and IL-18 levels were found to be elevated in THP-1 cells transduced with NLRP3-Q705K compared to the NLRP3-WT. Upon exposure to alum, THP-1 cells stably expressing NLRP3-Q705K displayed an increased release of IL-1β, IL-18 and TNF-α, in a caspase-1 and IL-1 receptor-dependent manner.

Conclusions

Collectively, these findings show that the Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to an overactive NLRP3 inflammasome. The option of IL-1β blockade may be considered in patients with chronic inflammatory disorders that are unresponsive to conventional treatments.