APMIS 123: 163–168
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12333
TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis ANDREANA MARINO,1 SIMONA PERGOLIZZI,2 EUGENIA R. LAURIANO,2 GIUSEPPE SANTORO,3 FRANCESCA SPATARO,1 FRANCESCO CIMINO,1 ANTONIO SPECIALE,1 ANTONIA NOSTRO1 and GIUSEPPE BISIGNANO1 1
Department of Pharmaceutical Sciences and Health Products, University of Messina, Polo Annunziata, Messina; 2Department of Environmental Science, Territorial, Food and Health Security, University of Messina, Messina; and 3Department of Biomedical Sciences and of Morphological and Functional Images, A.O.U. Policlinic ‘G. Martino’, University of Messina, Messina, Italy
Marino A, Pergolizzi S, Lauriano ER, Santoro G, Spataro F, Cimino F, Speciale A, Nostro A, Bisignano G. TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis. APMIS 2015; 123: 163–168. The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences. Key words: Keratocytes; myofibroblasts; gram-positive; innate immunity; corneal culture. Giuseppe Santoro, Department of Biomedical Sciences and of Morphological and Functional Images, A.O.U. Policlinic ‘G. Martino’, University of Messina, Via Consolare Valeria, Messina 98125, Italy. e-mail: [email protected]
The cornea is highly resistant to infection despite its constant exposure to a wide array of microorganisms (1). However, once the epithelial integrity is breached, pathogens may invade the stroma leading to microbial infections of the cornea, commonly termed infective keratitis, which often result in loss of vision (2, 3). Staphylococcus aureus (S. aureus) is among the leading causes of keratitis that occurs mainly in contact lens wearers and those with corneal injury, with an increased number of isolates exhibiting antibiotic resistance (4–8). When S. aureus penetrates the corneal epithelium and the corneal stroma, there is rapid bacterial replication, production of toxins, including hemolytic a-toxin, and also stimulates extensive neutrophil infiltration
to the corneal stroma, and subsequent degranulation and release of cytotoxic mediators further contribute to the pathogenesis of keratitis (9–12). Damage to the corneal epithelium releases proinflammatory cytokines such as interleukin-1, transforming growth factor-b (TGFb) and plateletderived growth factor (PDGF) (13) to activate stromal keratocytes into myofibroblasts at the site of injury (14, 15), thus resulting in wound contraction and reorganization of extracellular matrix in the corneal stroma (16, 17). The ability of corneal cells to recognize and respond to microbial components is attributed largely to the family of Toll-like receptors (TLRs) (2, 18). Corneal epithelial cells express the TLR2 which recognizes and responds to S. aureus infection by the expression and secretion of
Received 31 January 2014. Accepted 22 September 2014
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pro-inflammatory cytokines (Il-6, IL-8) in a TLR2/ MyD88 dependent manner (10, 19, 20). In the current study, an ex vivo rabbit model of keratitis induced by S. aureus has been used to examine the TLR2 expression of corneal stromal cells. MATERIALS AND METHODS S. aureus strains and preparation Overnight cultures of S. aureus ATCC 6538P and S. aureus ATCC 29213 in Tryptic Soy Broth (TSB; Oxoid, Basingstoke, UK) at 37°C were washed three times with phosphate-buffered saline (PBS, pH 7.4) and then diluted to an optical density of 0.3 at 650 nm (approximately 5 9 108 CFU/mL). A UV germicidal lamp was utilized to inactivate S. aureus strains for 15 min. Bacterial killing was confirmed by incubating treated bacteria on blood agar plates.
TLR2 stimulation Normal rabbit eyes, obtained from a local abattoir, were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Ham’s F-12, Gibco, UK) with antibiotic/ antimycotic solution (1:200). To prepare the corneal culture, the sclero-corneal ring of 36 eyes were placed on the organ support in the dishes containing the tissue culture medium, as previously reported (20). The rings then, randomly, were divided into two groups of eighteen eyes each as follows: group 1– epithelial abrasion: a demarcated central area of the epithelial layer was abraded with a surgical trephine; group 2 – stromal abrasion: to simulate a deep corneal wound, the full-thickness epithelium was mechanically removed by scraping procedure always using the surgical trephine in order to expose the stromal surface. The first group was then divided into three sub-groups: 1a. Epithelial abrasion exposed to 4 lL of S. aureus ATCC 6538P 5 9 108 number of bacteria mL 1. 1b. Epithelial abrasion exposed to 4 lL of S. aureus ATCC 29213 5 9 108 number of bacteria mL 1. 1c. Epithelial abrasion control. whereas, the second group was divided in: 2a. Stromal abrasion exposed to 4 lL of S. aureus ATCC 6538P 5 9 108 number of bacteria mL 1. 2b. Stromal abrasion exposed to 4 lL of S. aureus ATCC 29213 5 9 108 number of bacteria mL 1. 2c. Stromal abrasion control. The organ cultures were incubated at 37°C in a humidified atmosphere of 6% CO2 for 48 h. The culture medium in the dishes was changed once during the experiment (each 24 h).
Quantitative RT-PCR RNA was extracted from stromal cells and TLR2 and IL8 mRNA levels were evaluated by Real-time PCR. Considering that corneal epithelial cells renewal is greatly
164
increased during wound healing (21), at 48 h in culture, the uppermost thin renovated superficial epithelial layers of all epithelial abrasion sub-groups (3 eyes per group) were scraped off and the stromal cells were removed by using a 13 mm pre-autoclaved membrane filter (Millipore, Milan, Italy). At 48 h in culture the cells of all stromal abrasion sub-groups (3 eyes per group) too were removed by using the membrane filter above mentioned. TLR2 and IL8 genes expression was measured as previously described (20). All the experiments were performed in duplicate and repeated three times. Results are expressed as means SD from two experiments and statistically analyzed by a one-way ANOVA test, followed by Tukey’s HSD. Differences in groups and treatments were considered significant for p < 0.05.
Confocal laser scanning microscopy analysis To visualize the expression of TLR2 in stromal cells, immunohistochemical examination was performed using confocal image analysis. The cornea samples (3 eyes per group) were treated at 48 h as previously described (20). Primary antibodies - a-smooth muscle actin (a-SMA) (Sigma-Aldrich, Milan, Italy; 5 lg mL 1) and TLR2 (Active Motif, Vinci-Biochem, Florence, Italy; 1:125) – and secondary antibodies – donkey anti-mouse IgG FITC conjugated and donkey anti-rabbit IgG TRITC conjugated (Alexa Fluorâ 488, 1:100; Alexa Fluorâ 594, 1:100; Invitrogen, Life Technologies, Monza, Italy) – were used. After washing, the sections were mounted with ProLongâ Gold Antifade Reagent with DAPI (Invitrogen) to prevent photobleaching, and coverslipped. Control experiments were performed in which the primary antibody was excluded. The sections were analyzed and images acquired using a Zeiss LSM 5 DUO confocal laser scanning microscope (Carl Zeiss, Jena, Germany). For each microscopic field, the number of a-SMA+ and TLR2+ cells was counted. Staining was evaluated independently by two different investigators. The results were expressed as the percentage of positive cells as mean value from several experiments.
RESULTS AND DISCUSSION The results demonstrated that TLR2 mRNA and IL-8 mRNA were highly expressed in the stromal cells of the groups exposed to S. aureus strains. In epithelial abrasion groups (1 group), activated stromal cells expressed higher TLR2 mRNA and IL-8 mRNA levels for S. aureus ATCC 6538P-1a group and S. aureus ATCC 29213-1b group, when compared to stromal control cells (1c group) (Fig. 1). In stromal abrasion groups (2 group), the TLR2 mRNA levels for S. aureus ATCC 6538P-2a group and S. aureus ATCC 29213-2b group as also IL8 mRNA levels for S. aureus ATCC 6538P-2a group and S. aureus ATCC 29213-2b group, were significantly higher in activated stromal cells when compared to stromal control cells (2c group) (Fig. 2). The higher levels of TLR2 and Il-8 mRNA, © 2014 APMIS. Published by John Wiley & Sons Ltd
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Fig. 1. TLR2 and IL-8 mRNA levels in stromal cells exposed to inactivated S. aureus (S.a.) strains after epithelial abrasion (1a–1b groups) compared with those of stromal cells not exposed to bacteria (1c control group). Values are expressed # as 2 DDCt normalized to control. *p < 0.05 vs respective control, p < 0.05 vs inactivated S. aureus ATCC
29213.
Fig. 2. TLR2 and IL-8 mRNA levels in stromal cells exposed to inactivated S. aureus (S.a.) strains after stromal abrasion (2a–2b groups) compared with those of stromal cells not exposed to bacteria (2c control group). Values are expressed as 2 DDCt normalized to control. *p < 0.05 vs respective control, #p < 0.05 vs inactivated S. aureus ATCC 29213.
obtained after stromal abrasion, could be due to the greater number of cells directly exposed to S. aureus compared to those activated by S. aureus penetrated into the subjacent stroma after epithelial abrasion. The immunofluorescence showed that keratocytes differentiated into myofibroblasts (green positive reaction for alpha-smooth muscle actin) after stromal abrasion, and 85% of them became capable of expressing TLR2 (red positive reaction) only when exposed to S. aureus (Fig. 3 A, B, C). On the contrary, the control samples demonstrated the activation of myofibroblasts unable to express the TLR2 (Fig. 3D). The same immunofluorescence pattern of a-SMA and TLR2 was observed in myofibroblasts of the epithelial abrasion group, even if only 33% © 2014 APMIS. Published by John Wiley & Sons Ltd
of cells were able to express TLR2 (data not shown). Physical or chemical injury to the corneal surface can compromise the integrity of the epithelial barrier and allow entry of live organisms and microbial products to underlying epithelium and to the stroma, where they can induce an inflammatory response that can lead to visual impairment and possibly blindness (22). S. aureus has a formidable repertoire of virulence factors: cell-surface proteins and numerous secreted virulence factors contribute to the development of infections by promoting bacterial adhesion, circumventing host immune defenses and causing cell or tissue damage (11). Virulence factors are crucial for development of staphylococcal infections; however, host-related determinants,
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A
B
C
D
Fig. 3. Confocal laser scanning microscope study in myofibroblasts exposed (A, B, C) or not exposed (D) to inactivated S. aureus after stromal abrasion. Double-localization reactions using antibodies against a-SMA (green channel) and TLR2 (red channel) were performed. A: the keratocytes, differentiated into myofibroblasts, show an evident positive reaction for a- SMA; B: the stromal cells express the TLR2; C: the yellow fluorescence, in the merged image, confirms the presence of TLR2 in myofibroblasts due to an overlap of green and red fluorescence. The insert of A, B, C: detail of myofibroblast (Scale bar: 10 l); D: the keratocytes not exposed to inactivated S. aureus, differentiate into myofibroblasts but are unable to express the TLR2. Scale bar, 20 l.
such as immune competence, also play an essential role in infection (23). This immune competence represents the early host defense against microbial infection and involves several resident and immune cells (i.e. antigen presenting cells, dendritic cells and natural killer cells), chemokines and cytokines (18). Given that the major function of TLRs is pathogen recognition, it follows that these receptors play an important role in the ocular surface immune response to various agents (24). In the current study, we have shown the response of corneal stromal cells to S. aureus after epithelial or stromal injury. The increased expression of TLR2 and IL-8 of stromal cells showed the immediate innate response specific to S. aureus stimulation. Moreover, we have demonstrated, for the first time, that keratocytes, corneal residential stromal cells, differentiated into myofibroblasts after corneal injury became able to express TLR2 only when exposed to S. aureus. Keratocytes, the first component of the stroma to contact ocular pathogens when the epithelial barrier breaks down, differentiate into myofibro166
blast phenotype that became able to express alpha-smooth muscle actin (a-SMA), a contractile cytoskeletal element organized in bundles called stress fibers, able to exert contractile forces important to ensure rapid closure of the wound and surface re-epithelialization (25–28). During microbial infection, wound corneal fibroblasts differentiate into contractile myofibroblasts constitutively able to express TLR2 to recognize bacteria and to participate in remodeling of the wound through phagocytosis in the initial reaction of the avascular corneal stroma (24, 29). TLRs are not phagocytic receptors per se but they are also internalized in the process and therefore participate in the link between phagocytosis and inflammatory responses by triggering the production of cytokines (30). Myofibroblasts have been shown able to engulf bacteria and other foreign bodies (26), so these phagocytic properties might be the reason why these cells expressed the TLR2 only when they were in contact with S. aureus. © 2014 APMIS. Published by John Wiley & Sons Ltd
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Our findings suggest that TLR2 may play an important role in promoting the clearance of bacteria through upregulation of myofibroblasts phagocytic activity and induction of chemokines production. In addition, we have previously demonstrated a highly significant increase in TLR2 distribution on the epithelium surface of the cells of the abraded corneas when exposed to S. aureus (20). Taken together, our results help to demonstrate that, in response to S. aureus, epithelial and stromal cells increase the expression of TLR2. The increased expression of TLR2 on epithelial cells surface supports the hypothesis that TLR2 may help to regulate immune responses by acting as the first line of defense while myofibroblasts by TLR2 may instead be involved in the second line of defense of this avascular tissue against invading organisms. Further studies analyzing signaling pathway are mandatory to validate exact mechanism of interaction between myofibroblasts and pathogens. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation especially after keratoplasty, trauma, and refractive surgery. Moreover, these results indicate that the combination of corneal organ culture and experimental bacterial keratitis has the potential to be used as a mechanistically based alternative to in vivo animal testing.
9. 10.
11. 12. 13.
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16. 17. 18.
REFERENCES 19. 1. Ladage PM. What does overnight lens wear do to the corneal epithelium?: is corneal refractive therapy different? Eye Contact Lens 2004;30:194–7. 2. Kumar A, Yu FS. Toll-like receptors and corneal innate immunity. Curr Mol Med 2006;6:327–37. 3. Yu FS, Hazlett LD. Toll-like receptors and the eye. Invest Ophthalmol Vis Sci 2006;47:1255–63. 4. Forster W, Becker K, Hungermann D, Busse H. Methicillin-resistant Staphylococcus aureus keratitis after excimer laser photorefractive keratectomy. J Cataract Refract Surg 2002;28:722–4. 5. Shanmuganathan VA, Armstrong M, Buller A, Tullo AB. External ocular infections due to methicillinresistant Staphylococcus aureus (MRSA). Eye 2005;19:284–91. 6. Heimer SR, Yamada A, Russell H, Gilmore MS. Response of corneal epithelial cells to Staphylococcus aureus. Virulence 2010;1:223–35. 7. Major JC Jr, Engelbert M, Flynn HW Jr, Miller D, Smiddy WE, Davis JL. Staphylococcus aureus endophthalmitis: antibiotic susceptibilities, methicillin resistance, and clinical outcomes. Am J Ophthalmol 2010;149:278–83. 8. Sotozono C, Fukuda M, Ohishi M, Yano K, Origasa H, Saiki Y, et al. Vancomycin ophthalmic ointment 1% for methicillin-resistant Staphylococcus aureus or
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methicillin-resistant Staphylococcus epidermidis infections: a case series. BMJ Open 2013;3:1–6. O’Callaghan RJ, Girgis DO, Dajcs JJ, Sloop GD. Host defense against bacterial keratitis. Ocul Immunol Inflamm 2003;11:171–81. Sun Y, Hise AG, Kalsow CM, Pearlman E. Staphylococcus aureus-induced corneal inflammation is dependent on Toll-like receptor 2 and myeloid differentiation factor 88. Infect Immun 2006;74:5325–32. Fournier B. The function of TLR2 during staphylococcal diseases. Front Cell Infect Microbiol 2013;2:1–8. Marquart ME, O’Callaghan RJ. Infectious Keratitis: secreted bacterial proteins that mediate corneal damage. J Ophthalmol 2013;2013:1–9. Gabison EE, Huet E, Baudouin C, Menashi S. Direct epithelial stromal interaction in corneal wound healing: role of EMMPRIN/CD147 in MMPs induction and beyond. Prog Retin Eye Res 2009;28:19–33. Jester JV, Barry-Lane PA, Cavanagh HD, Petroll WM. Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes. Cornea 1996;15:505–16. Beales MP, Funderburgh JL, Jester JV, Hassell JR. Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture. Invest Ophthalmol Vis Sci 1999;40:1658–63. Jester JV, Petroll M, Cavanagh D. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Prog Retin Eye Res 1999;18:311–56. Hinz B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol 2007;127:526– 37. Lambiase A, Micera A, Sacchetti M, Mantelli F, Bonini S. Toll-like receptors in ocular surface diseases: overview and new findings. Clin Sci (Lond) 2011;120:441–50. Dziarski R, Gupta D. Staphylococcus aureus peptidoglycan is a toll-like receptor 2 activator: a reevaluation. Infect Immun 2005;73:5212–6. Marino A, Santoro G, Spataro F, Lauriano ER, Pergolizzi S, Cimino F, et al. Resveratrol role in Staphylococcus aureus-induced corneal inflammation. Pathog Dis 2013;68:61–4. Levin MH, Verkman AS. Aquaporin-3-dependent cell migration and proliferation during corneal re-epithelialization. Invest Ophthalmol Vis Sci 2006;47:4365–72. Johnson AC, Heinzel FP, Diaconu E, Sun Y, Hise AG, Golenbock D, et al. Activation of Toll-like receptor (TLR) 2, TLR4, and TLR9 in the mammalian cornea induces MyD88-dependent corneal inflammation. Invest Ophthalmol Vis Sci 2005;46:589–95. Tong SY, Chen LF, Fowler VG Jr. Colonization, pathogenicity, host susceptibility, and therapeutics for Staphylococcus aureus: what is the clinical relevance? Semin Immunopathol 2012;34:185–200. Choi HJ, Kim MK, Ko JH, Lee HJ, Jeong HJ, Wee WR, et al. Effect of Toll-like receptor 2 and 4 of corneal fibroblast on cytochine expression with cocultured antigen presenting cells. Cytochine 2011;56: 265–71. Fini ME. Keratocyte and fibroblast phenotypes in the repairing cornea. Prog Retin Eye Res 1999;18:529–51.
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26. Ebihara N, Yamagami S, Chen L, Tokura T, Iwatsu M, Ushio H, et al. Expression and function of Toll-like Receptor-3 and -9 in human corneal myofibroblasts. Invest Ophthalmol Vis Sci 2007;48: 3069–76. 27. Chaurasia SS, Kaur H, Medeiros FW, Smith SD, Wilson SE. Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury. Exp Eye Res 2009;89:133–9.
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28. Myrna KE, Pot SA, Murphy CJ. Meet the corneal myofibroblast: the role of myofibroblasts transformation in corneal wound healing and pathology. Vet Ophthalmol 2009;12:25–7. 29. Blander JM, Medzhitov R. Regulation of phagosome maturation by signals from toll-like receptor. Science 2004;304:1014–8. 30. Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 2002;14:103–10.
© 2014 APMIS. Published by John Wiley & Sons Ltd
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12333
TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis ANDREANA MARINO,1 SIMONA PERGOLIZZI,2 EUGENIA R. LAURIANO,2 GIUSEPPE SANTORO,3 FRANCESCA SPATARO,1 FRANCESCO CIMINO,1 ANTONIO SPECIALE,1 ANTONIA NOSTRO1 and GIUSEPPE BISIGNANO1 1
Department of Pharmaceutical Sciences and Health Products, University of Messina, Polo Annunziata, Messina; 2Department of Environmental Science, Territorial, Food and Health Security, University of Messina, Messina; and 3Department of Biomedical Sciences and of Morphological and Functional Images, A.O.U. Policlinic ‘G. Martino’, University of Messina, Messina, Italy
Marino A, Pergolizzi S, Lauriano ER, Santoro G, Spataro F, Cimino F, Speciale A, Nostro A, Bisignano G. TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis. APMIS 2015; 123: 163–168. The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences. Key words: Keratocytes; myofibroblasts; gram-positive; innate immunity; corneal culture. Giuseppe Santoro, Department of Biomedical Sciences and of Morphological and Functional Images, A.O.U. Policlinic ‘G. Martino’, University of Messina, Via Consolare Valeria, Messina 98125, Italy. e-mail: [email protected]
The cornea is highly resistant to infection despite its constant exposure to a wide array of microorganisms (1). However, once the epithelial integrity is breached, pathogens may invade the stroma leading to microbial infections of the cornea, commonly termed infective keratitis, which often result in loss of vision (2, 3). Staphylococcus aureus (S. aureus) is among the leading causes of keratitis that occurs mainly in contact lens wearers and those with corneal injury, with an increased number of isolates exhibiting antibiotic resistance (4–8). When S. aureus penetrates the corneal epithelium and the corneal stroma, there is rapid bacterial replication, production of toxins, including hemolytic a-toxin, and also stimulates extensive neutrophil infiltration
to the corneal stroma, and subsequent degranulation and release of cytotoxic mediators further contribute to the pathogenesis of keratitis (9–12). Damage to the corneal epithelium releases proinflammatory cytokines such as interleukin-1, transforming growth factor-b (TGFb) and plateletderived growth factor (PDGF) (13) to activate stromal keratocytes into myofibroblasts at the site of injury (14, 15), thus resulting in wound contraction and reorganization of extracellular matrix in the corneal stroma (16, 17). The ability of corneal cells to recognize and respond to microbial components is attributed largely to the family of Toll-like receptors (TLRs) (2, 18). Corneal epithelial cells express the TLR2 which recognizes and responds to S. aureus infection by the expression and secretion of
Received 31 January 2014. Accepted 22 September 2014
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pro-inflammatory cytokines (Il-6, IL-8) in a TLR2/ MyD88 dependent manner (10, 19, 20). In the current study, an ex vivo rabbit model of keratitis induced by S. aureus has been used to examine the TLR2 expression of corneal stromal cells. MATERIALS AND METHODS S. aureus strains and preparation Overnight cultures of S. aureus ATCC 6538P and S. aureus ATCC 29213 in Tryptic Soy Broth (TSB; Oxoid, Basingstoke, UK) at 37°C were washed three times with phosphate-buffered saline (PBS, pH 7.4) and then diluted to an optical density of 0.3 at 650 nm (approximately 5 9 108 CFU/mL). A UV germicidal lamp was utilized to inactivate S. aureus strains for 15 min. Bacterial killing was confirmed by incubating treated bacteria on blood agar plates.
TLR2 stimulation Normal rabbit eyes, obtained from a local abattoir, were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Ham’s F-12, Gibco, UK) with antibiotic/ antimycotic solution (1:200). To prepare the corneal culture, the sclero-corneal ring of 36 eyes were placed on the organ support in the dishes containing the tissue culture medium, as previously reported (20). The rings then, randomly, were divided into two groups of eighteen eyes each as follows: group 1– epithelial abrasion: a demarcated central area of the epithelial layer was abraded with a surgical trephine; group 2 – stromal abrasion: to simulate a deep corneal wound, the full-thickness epithelium was mechanically removed by scraping procedure always using the surgical trephine in order to expose the stromal surface. The first group was then divided into three sub-groups: 1a. Epithelial abrasion exposed to 4 lL of S. aureus ATCC 6538P 5 9 108 number of bacteria mL 1. 1b. Epithelial abrasion exposed to 4 lL of S. aureus ATCC 29213 5 9 108 number of bacteria mL 1. 1c. Epithelial abrasion control. whereas, the second group was divided in: 2a. Stromal abrasion exposed to 4 lL of S. aureus ATCC 6538P 5 9 108 number of bacteria mL 1. 2b. Stromal abrasion exposed to 4 lL of S. aureus ATCC 29213 5 9 108 number of bacteria mL 1. 2c. Stromal abrasion control. The organ cultures were incubated at 37°C in a humidified atmosphere of 6% CO2 for 48 h. The culture medium in the dishes was changed once during the experiment (each 24 h).
Quantitative RT-PCR RNA was extracted from stromal cells and TLR2 and IL8 mRNA levels were evaluated by Real-time PCR. Considering that corneal epithelial cells renewal is greatly
164
increased during wound healing (21), at 48 h in culture, the uppermost thin renovated superficial epithelial layers of all epithelial abrasion sub-groups (3 eyes per group) were scraped off and the stromal cells were removed by using a 13 mm pre-autoclaved membrane filter (Millipore, Milan, Italy). At 48 h in culture the cells of all stromal abrasion sub-groups (3 eyes per group) too were removed by using the membrane filter above mentioned. TLR2 and IL8 genes expression was measured as previously described (20). All the experiments were performed in duplicate and repeated three times. Results are expressed as means SD from two experiments and statistically analyzed by a one-way ANOVA test, followed by Tukey’s HSD. Differences in groups and treatments were considered significant for p < 0.05.
Confocal laser scanning microscopy analysis To visualize the expression of TLR2 in stromal cells, immunohistochemical examination was performed using confocal image analysis. The cornea samples (3 eyes per group) were treated at 48 h as previously described (20). Primary antibodies - a-smooth muscle actin (a-SMA) (Sigma-Aldrich, Milan, Italy; 5 lg mL 1) and TLR2 (Active Motif, Vinci-Biochem, Florence, Italy; 1:125) – and secondary antibodies – donkey anti-mouse IgG FITC conjugated and donkey anti-rabbit IgG TRITC conjugated (Alexa Fluorâ 488, 1:100; Alexa Fluorâ 594, 1:100; Invitrogen, Life Technologies, Monza, Italy) – were used. After washing, the sections were mounted with ProLongâ Gold Antifade Reagent with DAPI (Invitrogen) to prevent photobleaching, and coverslipped. Control experiments were performed in which the primary antibody was excluded. The sections were analyzed and images acquired using a Zeiss LSM 5 DUO confocal laser scanning microscope (Carl Zeiss, Jena, Germany). For each microscopic field, the number of a-SMA+ and TLR2+ cells was counted. Staining was evaluated independently by two different investigators. The results were expressed as the percentage of positive cells as mean value from several experiments.
RESULTS AND DISCUSSION The results demonstrated that TLR2 mRNA and IL-8 mRNA were highly expressed in the stromal cells of the groups exposed to S. aureus strains. In epithelial abrasion groups (1 group), activated stromal cells expressed higher TLR2 mRNA and IL-8 mRNA levels for S. aureus ATCC 6538P-1a group and S. aureus ATCC 29213-1b group, when compared to stromal control cells (1c group) (Fig. 1). In stromal abrasion groups (2 group), the TLR2 mRNA levels for S. aureus ATCC 6538P-2a group and S. aureus ATCC 29213-2b group as also IL8 mRNA levels for S. aureus ATCC 6538P-2a group and S. aureus ATCC 29213-2b group, were significantly higher in activated stromal cells when compared to stromal control cells (2c group) (Fig. 2). The higher levels of TLR2 and Il-8 mRNA, © 2014 APMIS. Published by John Wiley & Sons Ltd
TLR2 IN CORNEAL STROMA BY S. AUREUS
Fig. 1. TLR2 and IL-8 mRNA levels in stromal cells exposed to inactivated S. aureus (S.a.) strains after epithelial abrasion (1a–1b groups) compared with those of stromal cells not exposed to bacteria (1c control group). Values are expressed # as 2 DDCt normalized to control. *p < 0.05 vs respective control, p < 0.05 vs inactivated S. aureus ATCC
29213.
Fig. 2. TLR2 and IL-8 mRNA levels in stromal cells exposed to inactivated S. aureus (S.a.) strains after stromal abrasion (2a–2b groups) compared with those of stromal cells not exposed to bacteria (2c control group). Values are expressed as 2 DDCt normalized to control. *p < 0.05 vs respective control, #p < 0.05 vs inactivated S. aureus ATCC 29213.
obtained after stromal abrasion, could be due to the greater number of cells directly exposed to S. aureus compared to those activated by S. aureus penetrated into the subjacent stroma after epithelial abrasion. The immunofluorescence showed that keratocytes differentiated into myofibroblasts (green positive reaction for alpha-smooth muscle actin) after stromal abrasion, and 85% of them became capable of expressing TLR2 (red positive reaction) only when exposed to S. aureus (Fig. 3 A, B, C). On the contrary, the control samples demonstrated the activation of myofibroblasts unable to express the TLR2 (Fig. 3D). The same immunofluorescence pattern of a-SMA and TLR2 was observed in myofibroblasts of the epithelial abrasion group, even if only 33% © 2014 APMIS. Published by John Wiley & Sons Ltd
of cells were able to express TLR2 (data not shown). Physical or chemical injury to the corneal surface can compromise the integrity of the epithelial barrier and allow entry of live organisms and microbial products to underlying epithelium and to the stroma, where they can induce an inflammatory response that can lead to visual impairment and possibly blindness (22). S. aureus has a formidable repertoire of virulence factors: cell-surface proteins and numerous secreted virulence factors contribute to the development of infections by promoting bacterial adhesion, circumventing host immune defenses and causing cell or tissue damage (11). Virulence factors are crucial for development of staphylococcal infections; however, host-related determinants,
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A
B
C
D
Fig. 3. Confocal laser scanning microscope study in myofibroblasts exposed (A, B, C) or not exposed (D) to inactivated S. aureus after stromal abrasion. Double-localization reactions using antibodies against a-SMA (green channel) and TLR2 (red channel) were performed. A: the keratocytes, differentiated into myofibroblasts, show an evident positive reaction for a- SMA; B: the stromal cells express the TLR2; C: the yellow fluorescence, in the merged image, confirms the presence of TLR2 in myofibroblasts due to an overlap of green and red fluorescence. The insert of A, B, C: detail of myofibroblast (Scale bar: 10 l); D: the keratocytes not exposed to inactivated S. aureus, differentiate into myofibroblasts but are unable to express the TLR2. Scale bar, 20 l.
such as immune competence, also play an essential role in infection (23). This immune competence represents the early host defense against microbial infection and involves several resident and immune cells (i.e. antigen presenting cells, dendritic cells and natural killer cells), chemokines and cytokines (18). Given that the major function of TLRs is pathogen recognition, it follows that these receptors play an important role in the ocular surface immune response to various agents (24). In the current study, we have shown the response of corneal stromal cells to S. aureus after epithelial or stromal injury. The increased expression of TLR2 and IL-8 of stromal cells showed the immediate innate response specific to S. aureus stimulation. Moreover, we have demonstrated, for the first time, that keratocytes, corneal residential stromal cells, differentiated into myofibroblasts after corneal injury became able to express TLR2 only when exposed to S. aureus. Keratocytes, the first component of the stroma to contact ocular pathogens when the epithelial barrier breaks down, differentiate into myofibro166
blast phenotype that became able to express alpha-smooth muscle actin (a-SMA), a contractile cytoskeletal element organized in bundles called stress fibers, able to exert contractile forces important to ensure rapid closure of the wound and surface re-epithelialization (25–28). During microbial infection, wound corneal fibroblasts differentiate into contractile myofibroblasts constitutively able to express TLR2 to recognize bacteria and to participate in remodeling of the wound through phagocytosis in the initial reaction of the avascular corneal stroma (24, 29). TLRs are not phagocytic receptors per se but they are also internalized in the process and therefore participate in the link between phagocytosis and inflammatory responses by triggering the production of cytokines (30). Myofibroblasts have been shown able to engulf bacteria and other foreign bodies (26), so these phagocytic properties might be the reason why these cells expressed the TLR2 only when they were in contact with S. aureus. © 2014 APMIS. Published by John Wiley & Sons Ltd
TLR2 IN CORNEAL STROMA BY S. AUREUS
Our findings suggest that TLR2 may play an important role in promoting the clearance of bacteria through upregulation of myofibroblasts phagocytic activity and induction of chemokines production. In addition, we have previously demonstrated a highly significant increase in TLR2 distribution on the epithelium surface of the cells of the abraded corneas when exposed to S. aureus (20). Taken together, our results help to demonstrate that, in response to S. aureus, epithelial and stromal cells increase the expression of TLR2. The increased expression of TLR2 on epithelial cells surface supports the hypothesis that TLR2 may help to regulate immune responses by acting as the first line of defense while myofibroblasts by TLR2 may instead be involved in the second line of defense of this avascular tissue against invading organisms. Further studies analyzing signaling pathway are mandatory to validate exact mechanism of interaction between myofibroblasts and pathogens. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation especially after keratoplasty, trauma, and refractive surgery. Moreover, these results indicate that the combination of corneal organ culture and experimental bacterial keratitis has the potential to be used as a mechanistically based alternative to in vivo animal testing.
9. 10.
11. 12. 13.
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16. 17. 18.
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