Srijata Sarkar

RNA-Binding Protein AUF1 Regulates Lipopolysaccharide-Induced IL10 Expression by Activating IκB Kinase Complex in Monocytes

ABSTRACT Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40AUF1 cDNA restores both signaling pathways. To define the molecular mechanisms by which p40AUF1 controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40AUF1 serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40AUF1 does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40AUF1 regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40(AUF1) cDNA restores both signaling pathways. To define the molecular mechanisms by which p40(AUF1) controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40(AUF1) serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40(AUF1) does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40(AUF1) regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.

Suppression of the NF-κB Pathway by Diesel Exhaust Particles Impairs Human Antimycobacterial Immunity

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1β, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.

RNA-binding Protein AUF1 Regulates LPS-induced IL10 Expression by Activating IκB Kinase Complex in Monocytes

ABSTRACT Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40AUF1 cDNA restores both signaling pathways. To define the molecular mechanisms by which p40AUF1 controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40AUF1 serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40AUF1 does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40AUF1 regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40(AUF1) cDNA restores both signaling pathways. To define the molecular mechanisms by which p40(AUF1) controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40(AUF1) serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40(AUF1) does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40(AUF1) regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.

Air Pollution Particulate Matter Alters Antimycobacterial Respiratory Epithelium Innate Immunity

ABSTRACT Inhalation exposure to indoor air pollutants and cigarette smoke increases the risk of developing tuberculosis (TB). Whether exposure to ambient air pollution particulate matter (PM) alters protective human host immune responses against Mycobacterium tuberculosis has been little studied. Here, we examined the effect of PM from Iztapalapa, a municipality of Mexico City, with aerodynamic diameters below 2.5 μm (PM2.5) and 10 μm (PM10) on innate antimycobacterial immune responses in human alveolar type II epithelial cells of the A549 cell line. Exposure to PM2.5 or PM10 deregulated the ability of the A549 cells to express the antimicrobial peptides human β-defensin 2 (HBD-2) and HBD-3 upon infection with M. tuberculosis and increased intracellular M. tuberculosis growth (as measured by CFU count). The observed modulation of antibacterial responsiveness by PM exposure was associated with the induction of senescence in PM-exposed A549 cells and was unrelated to PM-mediated loss of cell viability. Thus, the induction of senescence and downregulation of HBD-2 and HBD-3 expression in respiratory PM-exposed epithelial cells leading to enhanced M. tuberculosis growth represent mechanisms by which exposure to air pollution PM may increase the risk of M. tuberculosis infection and the development of TB.

Low-dose AgNPs reduce lung mechanical function and innate immune defense in the absence of cellular toxicity

Abstract Multiple studies have examined the direct cellular toxicity of silver nanoparticles (AgNPs). However, the lung is a complex biological system with multiple cell types and a lipid-rich surface fluid; therefore, organ level responses may not depend on direct cellular toxicity. We hypothesized that interaction with the lung lining is a critical determinant of organ level responses. Here, we have examined the effects of low dose intratracheal instillation of AgNPs (0.05 μg/g body weight) 20 and 110 nm diameter in size, and functionalized with citrate or polyvinylpyrrolidone. Both size and functionalization were significant factors in particle aggregation and lipid interaction in vitro. One day post-intratracheal instillation lung function was assessed, and bronchoalveolar lavage (BAL) and lung tissue collected. There were no signs of overt inflammation. There was no change in surfactant protein-B content in the BAL but there was loss of surfactant protein-D with polyvinylpyrrolidone (PVP)-stabilized particles. Mechanical impedance data demonstrated a significant increase in pulmonary elastance as compared to control, greatest with 110 nm PVP-stabilized particles. Seven days post-instillation of PVP-stabilized particles increased BAL cell counts, and reduced lung function was observed. These changes resolved by 21 days. Hence, AgNP-mediated alterations in the lung lining and mechanical function resolve by 21 days. Larger particles and PVP stabilization produce the largest disruptions. These studies demonstrate that low dose AgNPs elicit deficits in both mechanical and innate immune defense function, suggesting that organ level toxicity should be considered.

Modulation of Human Macrophage Responses to Mycobacterium tuberculosis by Silver Nanoparticles of Different Size and Surface Modification

Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.

Modeling in vitro cellular responses to silver nanoparticles.

Engineered nanoparticles (NPs) have been widely demonstrated to induce toxic effects to various cell types. In vitro cell exposure systems have high potential for reliable, high throughput screening of nanoparticle toxicity, allowing focusing on particular pathways while excluding unwanted effects due to other cells or tissue dosimetry. The work presented here involves a detailed biologically based computational model of cellular interactions with NPs; it utilizes measurements performed in human cell culture systems in vitro, to develop a mechanistic mathematical model that can support analysis and prediction of in vivo effects of NPs. The model considers basic cellular mechanisms including proliferation, apoptosis, and production of cytokines in response to NPs. This new model is implemented for macrophages and parameterized using in vitro measurements of changes in cellular viability and mRNA levels of cytokines: TNF, IL-1b, IL-6, IL-8, and IL-10. The model includes in vitro cellular dosimetry due to nanoparticle transport and transformation. Furthermore, the model developed here optimizes the essential cellular parameters based on in vitro measurements, and provides a “stepping stone” for the development of more advanced in vivo models that will incorporate additional cellular and NP interactions.

Variability in bioreactivity linked to changes in size and zeta potential of diesel exhaust particles in human immune cells

Acting as fuel combustion catalysts to increase fuel economy, cerium dioxide (ceria, CeO2) nanoparticles have been used in Europe as diesel fuel additives (Envirox™). We attempted to examine the effects of particles emitted from a diesel engine burning either diesel (diesel exhaust particles, DEP) or diesel doped with various concentrations of CeO2 (DEP-Env) on innate immune responses in THP-1 and primary human peripheral blood mononuclear cells (PBMC). Batches of DEP and DEP-Env were obtained on three separate occasions using identical collection and extraction protocols with the aim of determining the reproducibility of particles generated at different times. However, we observed significant differences in size and surface charge (zeta potential) of the DEP and DEP-Env across the three batches. We also observed that exposure of THP-1 cells and PBMC to identical concentrations of DEP and DEP-Env from the three batches resulted in statistically significant differences in bioreactivity as determined by IL-1β, TNF-α, IL-6, IFN-γ, and IL-12p40 mRNA (by qRT-PCR) and protein expression (by ELISPOT assays). Importantly, bioreactivity was noted in very tight ranges of DEP size (60 to 120 nm) and zeta potential (−37 to −41 mV). Thus, these physical properties of DEP and DEP-Env were found to be the primary determinants of the bioreactivity measured in this study. Our findings also point to the potential risk of over- or under- estimation of expected bioreactivity effects (and by inference of public health risks) from bulk DEP use without taking into account potential batch-to-batch variations in physical (and possibly chemical) properties.

Effect of inhaled air pollution particulate matter in alveolar macrophages on local pro-inflammatory cytokine and peripheral interferon γ production in response to Mycobacterium tuberculosis

Abstract Background Human alveolar macrophages are first-line cellular responders to inhaled particulate matter (PM) and microbial pathogens in the lungs. We examined whether PM exposure affects human bronchoalveolar and peripheral blood mononuclear cell responses to Mycobacterium tuberculosis . Methods Bronchoalveolar and peripheral blood mononuclear cells were obtained from 30 healthy adult volunteers living in Mexico City, Mexico. Proportions of alveolar macrophages with PM and areas of cytoplasm occupied by inhaled PM within alveolar macrophages were evaluated in 50 bronchoalveolar cells from each donor by ImageJ (version 1.49) on 1000× digital bright field microscopy cytospin images. Ambient PM 2.5 (aerodynamic diameters M tuberculosis (strain H37Ra at multiplicities of infection [MOI] of 1 and 10), lipopolysaccharide, 6 kDa early secretory antigenic target (ESAT-6), purified protein derivative, and PM 2.5 were enumerated by ELISPOT assays. Findings While 26–60% of bronchoalveolar cells harboured inhaled PM, percentages of alveolar macrophage areas covered by PM were between 0·9% (±0·16) and 5·49% (±0·78). In unstimulated bronchoalveolar cells and bronchoalveolar cells exposed in vitro to PM (PM 2.5 ; 0·1, 1·0, 10 μg/mL), frequencies of interleukin-1β and TNF-α-producing cells decreased with higher percentages of alveolar macrophages containing inhaled PM (r=–0·78, p=0·0078 and r=–0·63, p=0·038, respectively). By contrast, frequencies of interferon-γ-producing cells increased in bronchoalveolar cells with alveolar macrophages containing greater areas of inhaled PM (r=0·59, p=0·049). In bronchoalveolar cells infected in vitro with M tuberculosis , frequencies of cells producing interleukin 1β significantly decreased in volunteers with higher percentages of alveolar macrophages containing inhaled PM. Frequencies of interferon-γ-producing cells were significantly decreased in peripheral blood mononuclear cells stimulated with purified protein derivative from volunteers with higher percentages of inhaled PM-containing alveolar macrophages (r=–0·7167, p=0·0184). Interpretation The presence of inhaled PM in alveolar macrophages significantly decreases interleukin 1β and TNF-α production in bronchoalveolar cells in response to M tuberculosis infection as well as interferon-γ-production in peripheral blood mononuclear cells in response to purified protein derivative. These findings provide important insights into the potential mechanisms underlying the epidemiological observations of increased risk of tuberculosis development in air pollution exposure settings and indicate that air pollution may have an adverse impact on global tuberculosis control efforts. Funding NIEHS 5RO1ES020382-05.

Exposure to Silver Nanospheres Leads to Altered Respiratory Mechanics and Delayed Immune Response in an in Vivo Murine Model

Here we examine the organ level toxicology of both carbon black (CB) and silver nanoparticles (AgNP). We aim to determine metal-specific effects to respiratory function, inflammation and potential interactions with lung lining fluid (LLF). C57Bl6/J male mice were intratracheally instilled with saline (control), low (0.05 μg/g) or high (0.5 μg/g) doses of either AgNP or CB 15 nm nanospheres. Lung histology, cytology, surfactant composition and function, inflammatory gene expression, and pulmonary function were measured at 1, 3, and 7 days post-exposure. Acutely, high dose CB resulted in an inflammatory response, increased neutrophilia and cytokine production, without alteration in surfactant composition or respiratory mechanics. Low dose CB had no effect. Neither low nor high dose AgNPs resulted in an acute inflammatory response, but there was an increase in work of breathing. Three days post-exposure with CB, a persistent neutrophilia was noted. High dose AgNP resulted in an elevated number of macrophages and invasion of lymphocytes. Additionally, AgNP treated mice displayed increased expression of IL1B, IL6, CCL2, and IL10. However, there were no significant changes in respiratory mechanics. At day 7, inflammation had resolved in AgNP-treated mice, but tissue stiffness and resistance were significantly decreased, which was accompanied by an increase in surfactant protein D (SP-D) content. These data demonstrate that the presence of metal alters the response of the lung to nanoparticle exposure. AgNP-surfactant interactions may alter respiratory function and result in a delayed immune response, potentially due to modified airway epithelial cell function.