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Tiffany Hughes,
Brian Becknell,
Aharon G Freud,
Susan McClory,
Edward Briercheck,
Jianhua Yu,
Charlene Mao,
Chiara Giovenzana,
Gerard Nuovo,
Lai Wei,
Xiaoli Zhang,
Mikhail A Gavrilin, Mark D Wewers,
Michael A Caligiuri
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Mikhail A Gavrilin,
Dalia H A Abdelaziz,
Mahmoud Mostafa,
Basant A Abdulrahman,
Jaykumar Grandhi,
Anwari Akhter,
Arwa Abu Khweek,
Daniel F Aubert,
Miguel A Valvano, Mark D Wewers,
Amal O Amer
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Ruairi J Fahy,
Matthew C Exline,
Mikhail A Gavrilin,
Nitin Y Bhatt,
Beth Y Besecker,
Anasuya Sarkar,
Jennifer L Hollyfield,
Michelle D Duncan,
Haikady N Nagaraja,
Nina L Knatz,
Mark Hall, Mark D Wewers
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Ruairi J Fahy,
Matthew C Exline,
Mikhail A Gavrilin,
Nitin Y Bhatt,
Beth Y Besecker,
Anasuya Sarkar,
Jennifer L Hollyfield,
Michelle D Duncan,
Haikady N Nagaraja,
Nina L Knatz,
Mark Hall, Mark D Wewers
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Mikhail A Gavrilin,
Dalia H A Abdelaziz,
Mahmoud Mostafa,
Basant A Abdulrahman,
Jaykumar Grandhi,
Anwari Akhter,
Arwa Abu Khweek,
Daniel F Aubert,
Miguel A Valvano, Mark D Wewers,
Amal O Amer
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Ruairi J Fahy,
Matthew C Exline,
Mikhail A Gavrilin,
Nitin Y Bhatt,
Beth Y Besecker,
Anasuya Sarkar,
Jennifer L Hollyfield,
Michelle D Duncan,
Haikady N Nagaraja,
Nina L Knatz,
Mark Hall, Mark D Wewers
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Mikhail A Gavrilin,
Dalia H A Abdelaziz,
Mahmoud Mostafa,
Basant A Abdulrahman,
Jaykumar Grandhi,
Anwari Akhter,
Arwa Abu Khweek,
Daniel F Aubert,
Miguel A Valvano, Mark D Wewers,
Amal O Amer
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Anwari Akhter,
Kyle Caution,
Arwa Abu Khweek,
Mia Tazi,
Basant A Abdulrahman,
Dalia H A Abdelaziz,
Oliver H Voss,
Andrea I Doseff,
Hoda Hassan,
Abul K Azad,
Larry S Schlesinger, Mark D Wewers,
Mikhail A Gavrilin,
Amal O Amer
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Basant A Abdulrahman,
Arwa Abu Khweek,
Anwari Akhter,
Kyle Caution,
Sheetal Kotrange,
Dalia H A Abdelaziz,
Christie Newland,
Roberto Rosales-Reyes,
Benjamin Kopp,
Karen McCoy,
Richard Montione,
Larry S Schlesinger,
Mikhail A Gavrilin, Mark D Wewers,
Miguel A Valvano,
Amal O Amer
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Devyn D Gillette,
Prexy A Shah,
Thomas Cremer,
Mikhail A Gavrilin,
Beth Y Besecker,
Anasuya Sarkar,
Daren L Knoell,
Estelle Cormet-Boyaka, Mark D Wewers,
Jonathan P Butchar,
Susheela Tridandapani
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ABSTRACT: Burkholderia cenocepacia (B.c.), the causative agent of Cepacia Syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B.c. have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B.c. infection. Infection led to early signaling events such as activation of Erk, Akt and NF-κB. Further, TNFα, IL-6, IL-8 and IL-1β were all significantly induced upon infection, but no IL-1β was detected in the supernatants. Because caspase-1 is required for IL-1β processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1β following B.c. infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1β secretion.
Journal of Biological Chemistry 12/2012; · 4.77 Impact Factor
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ABSTRACT: Pyroptosis is a type of cell death in which danger associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) induce mononuclear phagocytes to activate caspase-1 and release mature IL-1β. Because the tyrosine kinase inhibitor AG126 can prevent DAMP/PAMP induced activation of caspase-1, we hypothesized that tipping the tyrosine kinase/phosphatase balance toward phosphorylation would promote caspase-1 activation and cell death. THP-1 derived macrophages were therefore treated with the potent specific tyrosine phosphatase inhibitor, sodium orthovanadate (OVN) and analyzed for caspase-1 activation and cell death. OVN induced generalized increase in phosphorylated proteins, IL-1β release and cell death in a time and dose dependent pattern. This OVN induced pyroptosis correlated with speck formations that contained the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Culturing the cells in the presence of extracellular K(+) (known to inhibit ATP dependent pyroptosis), a caspase inhibitor (ZVAD) or down regulating the expression of ASC with stable expression of siASC prevented the OVN induced pyroptosis. These data demonstrate that pyroptotic death is linked to tyrosine phosphatase activity providing novel targets for future pharmacologic interventions.
Biochemical and Biophysical Research Communications 07/2012; 425(2):384-9. · 2.48 Impact Factor
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Anwari Akhter,
Kyle Caution,
Arwa Abu Khweek,
Mia Tazi,
Basant A Abdulrahman,
Dalia H A Abdelaziz,
Oliver H Voss,
Andrea I Doseff,
Hoda Hassan,
Abul K Azad,
Larry S Schlesinger, Mark D Wewers,
Mikhail A Gavrilin,
Amal O Amer
[show abstract]
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ABSTRACT: Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.
Immunity 05/2012; 37(1):35-47. · 21.64 Impact Factor
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ABSTRACT: Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.
Journal of Biological Chemistry 05/2012; 287(28):23479-88. · 4.77 Impact Factor
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ABSTRACT: Macrophages are unique innate immune cells that play an integral role in the defense of the host by virtue of their ability
to recognize, engulf, and kill pathogens while sending out danger signals via cytokines to recruit and activate inflammatory
cells. It is becoming increasingly clear that purinergic signaling events are essential components of the macrophage response
to pathogen challenges and disorders such as sepsis may be, at least in part, regulated by these important sensors. The activation
of the P2X7 receptor is a powerful event in the regulation of the caspase-1 inflammasome. We provide evidence that the inflammasome activation
requires “priming” of macrophages prior to ATP activation of the P2X7R. Inhibition of the inflammasome activation by the tyrosine kinase inhibitor, AG126, suggests regulation by phosphorylation.
Finally, the P2X7R may also be activated by other elements of the host response such as the antimicrobial peptide LL-37, which adds a new,
physiologically relevant agonist to the P2X7R pathway. Therapeutic approaches to inflammation and sepsis will certainly be enhanced by an increased understanding of how
purinergic receptors modulate the inflammasomes.
Purinergic Signalling 04/2012; 5(2):189-195. · 3.16 Impact Factor
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Mikhail A Gavrilin,
Dalia H A Abdelaziz,
Mahmoud Mostafa,
Basant A Abdulrahman,
Jaykumar Grandhi,
Anwari Akhter,
Arwa Abu Khweek,
Daniel F Aubert,
Miguel A Valvano, Mark D Wewers,
Amal O Amer
[show abstract]
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ABSTRACT: Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.
The Journal of Immunology 02/2012; 188(7):3469-77. · 5.79 Impact Factor
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Hannah E Cummings,
Joseph Barbi,
Patrick Reville,
Steve Oghumu,
Nicholas Zorko,
Anasuya Sarkar,
Tracy L Keiser,
Bao Lu,
Thomas Rückle,
Sanjay Varikuti,
Claudio Lezama-Davila, Mark D Wewers,
Caroline Whitacre,
Danuta Radzioch,
Christian Rommel,
Stéphanie Seveau,
Abhay R Satoskar
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ABSTRACT: Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.
Proceedings of the National Academy of Sciences 01/2012; 109(4):1251-6. · 9.68 Impact Factor
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Sainath R Kotha,
Melissa G Piper,
Rishi B Patel,
Sean Sliman,
Smitha Malireddy,
Lingying Zhao,
Christopher P Baran,
Patrick S Nana-Sinkam, Mark D Wewers,
Debra Romberger,
Clay B Marsh,
Narasimham L Parinandi
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ABSTRACT: The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A(2) (cPLA(2)) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA(2) in A549 cells, which was attenuated by AACOCF(3) (cPLA(2) inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA(2) serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA(2). The poultry PM-induced cPLA(2) serine phosphorylation and IL-8 release were attenuated by AACOCF(3), PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA(2) knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA(2) activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.
Cell biochemistry and biophysics 12/2011; · 3.34 Impact Factor
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Basant A Abdulrahman,
Arwa Abu Khweek,
Anwari Akhter,
Kyle Caution,
Sheetal Kotrange,
Dalia H A Abdelaziz,
Christie Newland,
Roberto Rosales-Reyes,
Benjamin Kopp,
Karen McCoy,
Richard Montione,
Larry S Schlesinger,
Mikhail A Gavrilin, Mark D Wewers,
Miguel A Valvano,
Amal O Amer
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ABSTRACT: Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.
Autophagy 11/2011; 7(11):1359-70. · 7.45 Impact Factor
