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THESIS MAUSITA KARMAKAR 2014.pdf (8.11 MB)

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INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1BETA PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS

Karmakar, Mausita
http://rave.ohiolink.edu/etdc/view?acc_num=case1396544303

Abstract Details

2014, Doctor of Philosophy, Case Western Reserve University, Pathology.
Bacterial infections of the cornea caused by Pseudomonas aeruginosa, Streptococcus pneumoniae and Staphylococcus aureus are major causes of preventable blindness worldwide as indicated by the World Health Organization (WHO). The major risk factors for P. aeruginosa keratitis are contact lens wear and ocular injury, whereas S. pneumoniae infection primarily occurs following corneal trauma. Microbial infections are associated with a robust inflammatory response that includes infiltration of neutrophils and macrophages to the cornea, and results in corneal opacity and loss of vision. The series of studies described herein reveals an important role for neutrophils in producing IL-1beta during corneal infection by P. aeruginosa and S. pneumoniae and shows distinct pathways for IL-beta processing by these cells. In order to investigate the mechanism of IL-1beta; processing during P. aeruginosa keratitis, we established a murine model of corneal infection and demonstrated that neutrophils are the key cell type that produces IL-1beta early during infection (at 24hr). At this time point, there are significantly fewer infiltrating macrophages (approximately 3%) compared to neutrophils (approximately 20%) in the corneal stroma indicating a minimal contribution of macrophages in IL-beta processing. Additionally, we showed that IL-1beta processing in the cornea is independent of NLRC4 inflammasome and caspase-1. Using highly purified human and murine neutrophils, we demonstrated that blockade of neutrophil serine proteases like elastase and cathepsin G blocks IL-1beta processing and secretion. This was further supported by the observation that neutrophil elastase (NE)-/- mice have impaired bacterial killing and reduced IL-1beta in the cornea compared with C57BL/6 mice. Taken together, these findings indicate that during P. aeruginosa corneal infection, neutrophils are the predominant producers of IL-beta during early infection and that the processing relies on neutrophil elastase instead of NLRC4/caspase-1 inflammasome. In a similar model of murine S. pneumoniae corneal infection, we showed that unlike P. aeruginosa keratitis, IL-1beta processing by neutrophils is dependent on NLRP3/ASC inflammasome and caspase-1. This was confirmed using purified murine neutrophils, where IL-1beta secretion was found to be dependent on NLRP3, ASC and caspase-1. Confocal microscopy identified caspase-1 and NLRP3 specks in neutrophils upon inflammasome induction. Additionally, S. pneumoniae cytolytic toxin pneumolysin (Ply) was found to activate NLRP3 in neutrophils, as mutant strain (pneumolysin deficient bacteria) did not induce caspase-1 activation or IL-1beta secretion by neutrophils. We also demonstrated that Ply - induced pore formation leads to loss of intracellular K+, which is a sufficient and necessary to signal inflammasome activation in neutrophils. Blocking of pyroptotic cell death in neutrophils inhibited IL-beta; secretion but did not block loss of intracellular K+ in response to S. pneumoniae, indicating that loss of intracellular ion is a direct consequence of pore formation by Ply and not a secondary response of pyroptosis. In summary, these findings significantly advance our knowledge of neutrophil - mediated IL-1beta processing during infection with two major human bacterial pathogens. Since neutrophils are the first responders that infiltrate the tissue in the majority of microbial infections, these mechanisms of IL-1beta processing by neutrophils might also play an important role in other inflammatory conditions.
Eric Pearlman (Advisor)
George Dubyak (Committee Chair)
Amy G. Hise (Committee Member)
Arne Rietsch (Committee Member)
Brian A. Cobb (Committee Member)
Clive R. Hamlin (Committee Member)
Immunology; Ophthalmology; Pathology
neutrophil; IL1beta, inflammasome; NLRP3; ASC; Caspase-1; NLRC4; elastase; Pseudomoans aeruginosa; Streptococcus pneumoniae; keratitis

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Citations

  • Karmakar, M. (2014). INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1BETA PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1396544303

    APA Style (7th edition)

  • Karmakar, Mausita. INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1BETA PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS. 2014. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1396544303.

    MLA Style (8th edition)

  • Karmakar, Mausita. "INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1BETA PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS." Doctoral dissertation, Case Western Reserve University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1396544303

    Chicago Manual of Style (17th edition)

case1396544303
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© 2014, some rights reserved.Creative Commons License INFLAMMASOME DEPENDENT AND INDEPENDENT IL-1BETA PROCESSING BY NEUTROPHILS DURING BACTERIAL KERATITIS by Mausita Karmakar is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.