Examining the immune response of pneumolysin exposed human corneal epithelial cells in a keratitis model

Abstract. Host immune factors involved in bacterial ocular virulence were examined through the use of pneumolysin, a cytoplasmic protein and pneumococcal strain D39 utilizing human corneal epithelial cells (HCEC). Recombinant pneumolysin activity was determined by a hemolysis assay using the following concentrations: 50-1000 ng/mL. HCEC were grown to confluency and exposed to 103 to 107 colony forming units (CFUs) of mid-log phase Streptococcus pneumoniae D39 for 2, 4, 6, and 24 h. HCEC were exposed to pneumolysin at various concentrations: 200-1000 ng/mL. ELISA and human cytokine array was performed on the supernatant. Fluorescence studies using cytotoxicity assay were performed on the HCEC cells following infection to determine live versus dead cells. The hemolysis assay showed that a minimum of 100 ng/mL PLY will lyse the red blood cells. Cytokine array on the HCEC exposed supernatants showed the presence of IL-6 and IL-8. Four hour exposure of HCEC cells to a range of 103 to 107 CFU of D39 resulted in monolayer disruption and decreased viability as compared to cells exposed to the media alone. HCEC cells exposed to any of the concentrations of bacteria exhibited signs of cellular damage and death. Following 24 h exposure, there was an increased expression of IL-8 (p < 0.001) compared to media alone. IL-6, IL-8 and IFN-gamma levels were elicited at a minimal of 200ng PLY. In addition to changes in viability and morphology of cultured cells, we detected specific cytokines in the culture supernatants of HCEC following exposure to S. pneumoniae D39.

[1]  L. McDaniel,et al.  The Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae Binds to Lipid Raft Microdomains in Human Corneal Epithelial Cells , 2013, PloS one.

[2]  M. Marquart,et al.  Infectious Keratitis: Secreted Bacterial Proteins That Mediate Corneal Damage , 2013, Journal of ophthalmology.

[3]  R. Tweten,et al.  Membrane assembly of the cholesterol-dependent cytolysin pore complex. , 2012, Biochimica et biophysica acta.

[4]  Jacquelin Boggs.  Bacterial and Fungal Keratitis in the Equine Patient , 2011 .

[5]  William P. Hausdorff,et al.  Global epidemiology of pneumococcal disease--new prospects for vaccine control. , 2009, Advances in experimental medicine and biology.

[6]  Deborah A. Brown,et al.  Lipid rafts, detergent-resistant membranes, and raft targeting signals. , 2006, Physiology.

[7]  R. Tweten,et al.  Cholesterol-Dependent Cytolysins, a Family of Versatile Pore-Forming Toxins , 2005, Infection and Immunity.

[8]  I. Rouiller,et al.  Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy. , 2005, Journal of Structural Biology.

[9]  Zhifeng Shao,et al.  Vertical collapse of a cytolysin prepore moves its transmembrane β‐hairpins to the membrane , 2004, The EMBO journal.

[10]  R. Bals,et al.  Innate immunity in the lung: how epithelial cells fight against respiratory pathogens , 2004, European Respiratory Journal.

[11]  G. Gudmundsson,et al.  Respiratory Syncytial Virus Up-regulates TLR4 and Sensitizes Airway Epithelial Cells to Endotoxin* , 2003, Journal of Biological Chemistry.

[12]  F. Imani,et al.  Viral Induction of Inflammatory Cytokines in Human Epithelial Cells Follows a p38 Mitogen-Activated Protein Kinase-Dependent but NF-κB-Independent Pathway1 , 2003, The Journal of Immunology.

[13]  T. Pistole,et al.  Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine, IL-6. , 2002, Microbes and infection.

[14]  Ruslan Medzhitov,et al.  Toll-like receptors and innate immunity , 2001, Nature Reviews Immunology.

[15]  H. Taylor,et al.  Study of the First Contact Management Profile of Cases of Infectious Keratitis: A Hospital-Based Study , 2000, Cornea.

[16]  A. Ogunniyi,et al.  Comparative Virulence of Streptococcus pneumoniaeStrains with Insertion-Duplication, Point, and Deletion Mutations in the Pneumolysin Gene , 1999, Infection and Immunity.

[17]  S. Johnston,et al.  Low grade rhinovirus infection induces a prolonged release of IL-8 in pulmonary epithelium. , 1998, Journal of immunology.

[18]  P. Andrew,et al.  Amino acid changes affecting the activity of pneumolysin alter the behaviour of pneumococci in pneumonia. , 1998, Microbial pathogenesis.

[19]  S. Savkovic,et al.  Activation of NF-κB in intestinal epithelial cells by enteropathogenic Escherichia coli. , 1997, American journal of physiology. Cell physiology.

[20]  C. Janeway,et al.  A human homologue of the Drosophila Toll protein signals activation of adaptive immunity , 1997, Nature.

[21]  S. Savkovic,et al.  Activation of NFk B in intestinal epithelial cells by enteropathogenic Escherichia coli , 1997 .

[22]  P. McDonnell,et al.  The role of smears, cultures, and antibiotic sensitivity testing in the management of suspected infectious keratitis. , 1996, Ophthalmology.

[23]  P. McDonnell,et al.  The importance of initial management in the treatment of severe infectious corneal ulcers. , 1995, Ophthalmology.

[24]  J C Paton,et al.  Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae , 1995, Infection and immunity.

[25]  P. Andrew,et al.  Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae , 1994, Infection and immunity.

[26]  James M. Hill,et al.  Pharmacokinetic Considerations in the Treatment of Bacterial Keratitis , 1994, Clinical pharmacokinetics.

[27]  S. Normark,et al.  Molecular cross talk between epithelial cells and pathogenic microorganisms , 1991, Cell.

[28]  J. A. Hobden,et al.  The role of pneumolysin in ocular infections with Streptococcus pneumoniae. , 1990, Current eye research.

[29]  J. Alouf,et al.  Binding of cholesterol by sulfhydryl-activated cytolysins , 1980, Infection and immunity.