Epithelial cells of the cornea and the conjunctiva constitutively produce antimicrobial peptides; however, the production of defensins by other cell types located around the eye has not been investigated. We analyzed the production of beta-defensins (hBD) and cathelicidin LL-37 during the infection of primary limbo-corneal fibroblasts with M. tuberculosis (MTB), M. abscessus (MAB), and M. smegmatis (MSM). The intracellular survival of each mycobacterium, the production of cytokines and the changes on the distribution of the actin filaments during the infection were also analyzed. Fibroblasts produce basal levels of hBD1 and LL-37 and under PMA stimulation they produce hBD2, hBD3 and overexpress hBD1 and LL-37. MAB induced the highest levels of hBD1 and LL-37 and intermediate levels of IL-6; however, MAB was not eliminated. In addition, MAB induced the greatest change to the distribution of the actin filaments. MTB also produced changes in the structure of the cytoskeleton and induced low levels of hBD1 and IL-6, and intermediate levels of LL-37. The balance of these molecules induced by MTB appeared to contribute to the non-replicative state observed in the limbo-corneal cells. MSM induced the lowest levels of hBD1 and LL-37 but the highest levels of IL-6; MSM was eliminated. The results suggest that mycobacterial infections regulate the production of antimicrobial peptides and cytokines, which in conjunction can contribute to the control of the bacilli.
References
[1]
Sharma, S. Antibiotic resistance in ocular bacterial pathogens. Indian. J. Med. Microbiol. 2011, 29, 218–222, doi:10.4103/0255-0857.83903.
[2]
Ahmad, S. Pathogenesis, immunology and diagnosis of latent Mycobacterium tuberculosis infection. Clin. Dev. Immunol. 2011, 2011, 814–943.
H？fling-Lima, A.L.; de Freitas, D.; Sampaio, J.L.; Le？o, S.C.; Contarini, P. In vitro activity of fluoroquinolones against Mycobacterium abscessus and Mycobacterium chelonae causing infectious keratitis after LASIK in Brazil. Cornea 2005, 24, 730–734.
[5]
McNamara, N.A. Innate defense of the ocular surface. Eye Contact. Lens. 2003, 29, S10–S13, doi:10.1097/00140068-200301001-00004.
[6]
Qu, X.D.; Lehrer, R.I. Secretory phospholipase A2 is the principal bactericide for staphylococci and other Gram-positive bacteria in human tears. Infect. Immun. 1998, 66, 2791–2797.
[7]
Garreis, F.; Schlorf, T.; Worlitzsch, D.; Steven, P.; Br？uer, L.; J？ger, K.; Paulsen, F.P. Roles of human beta-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells. Histochem. Cell. Biol. 2010, 134, 59–73, doi:10.1007/s00418-010-0713-y.
[8]
Frohm, M.; Agerberth, B.; Ahangari, G.; Stahle-B？ckdahl, M.; Lidén, S.; Wigzell, H.; Gudmundsson, G.H. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. J. Biol. Chem. 1997, 272, 15258–15263.
[9]
Gropp, R.; Frye, M.; Wagner, T.O.; Bargon, J. Epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy. Hum. Gene. Ther. 1999, 10, 957–964, doi:10.1089/10430349950018355.
[10]
Harder, J.; Bartels, J.; Christophers, E.; Schr？der, J.M. A peptide antibiotic from human skin. Nature 1997, 387, 861.
[11]
Shin, D.M.; Jo, E.K. Antimicrobial Peptides in Innate Immunity against Mycobacteria. Immune. Netw. 2011, 11, 245–252.
Hoover, D.M.; Rajashankar, K.R.; Blumenthal, R.; Puri, A.; Oppenheim, J.J. Chertov, O.; Lubkowski, J. The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J. Biol. Chem. 2000, 275, 32911–32918.
[14]
Gordon, Y.J.; Huang, L.C.; Romanowski, E.G.; Yates, K.A.; Proske, R.J.; McDermott, A.M. Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity. Curr. Eye. Res. 2005, 30, 385–394.
[15]
Hattenbach, L.O.; Gümbel, H.; Kippenberger, S. Identification of beta-defensins in human conjunctiva. Antimicrob. Agents. Chemother. 1998, 42, 3332.
[16]
Haynes, R.J.; Tighe, P.J.; Dua, H.S. Antimicrobial defensin peptides of the human ocular surface. Br. J. Ophthalmol. 1999, 83, 737–741, doi:10.1136/bjo.83.6.737.
[17]
McDermott, A.M.; Redfern, R.L.; Zhang, B.; Pei, Y.; Huang, L.; Proske, R.J. Defensin expression by the cornea: multiple signaling pathways mediate IL-1 beta stimulation of hBD-2 expression by human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2003, 44, 1859–1865.
[18]
McNamara, N.A.; Van, R.; Tuchin, O.S.; Fleiszig, S.M. Ocular surface epithelia express mRNA for human beta defensin-2. Exp. Eye Res. 1999, 69, 483–490, doi:10.1006/exer.1999.0722.
[19]
Sack, R.A.; Conradi, L.; Krumholz, D.; Beaton, A.; Sathe, S.; Morris, C. Membrane array characterization of 80 chemokines, cytokines, and growth factors in open- and closed-eye tears: angiogenin and other defense system constituents. Invest. Ophthalmol. Vis. Sci. 2005, 46, 1228–1238, doi:10.1167/iovs.04-0760.
[20]
Huang, L.C.; Jean, D.; Proske, R.J.; Reins, R.Y.; McDermott, A.M. Ocular surface expression and in vitro activity of antimicrobial peptides. Curr. Eye Res. 2007, 32, 595–609, doi:10.1080/02713680701446653.
[21]
Haniffa, M.A.; Collin, M.P.; Buckley, C.D.; Dazzi, F. Mesenchymal stem cells: the fibroblasts’ new clothes? Haematologica 2009, 94, 258–263, doi:10.3324/haematol.13699.
[22]
Fries, K.M.; Blieden, T.; Looney, R.J.; Sempowski, G.D.; Silvera, M.R.; Willis, R.A.; Phipps, R.P. Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis. Clin. Immunol. Immunopathol. 1994, 72, 283–292.
[23]
Serhan, C.N.; Brain, S.D.; Buckley, C.D.; Gilroy, D.W.; Haslett, C.; O'Neill, L.A.; Perretti, M.; Rossi, A.G.; Wallace, J.L. Resolution of inflammation: state of the art, definitions and terms. FASEB J. 2007, 21, 325–332, doi:10.1096/fj.06-7227rev.
[24]
Le, J.M.; Vilcek, J. Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor. J. Immunol. 1987, 139, 3330–3337.
[25]
Shimabukuro, Y.; Murakami, S.; Okada, H. Interferon-gamma-dependent immunosuppressive effects of human gingival fibroblasts. Immunology 1992, 76, 344–347.
[26]
Strieter, R.M.; Gomperts, B.N.; Keane, M.P. The role of CXC chemokines in pulmonary fibrosis. J. Clin. Invest. 2007, 117, 549–556.
[27]
Fukuda, K. Role of corneal fibroblasts in the pathogenesis of ocular allergic diseases. Nippon. Ganka. Gakkai. Zasshi. 2005, 109, 717–726.
[28]
Sempowski, G.D.; Chess, P.R.; Moretti, A.J.; Padilla, J.; Phipps, R.P.; Blieden, T.M. CD40 mediated activation of gingival and periodontal ligament fibroblasts. J. Periodontol. 1997, 68, 284–292.
[29]
Hong, J.W.; Liu, J.J.; Lee, J.S.; Mohan, R.R.; Mohan, R.R.; Woods, D.J.; He, Y.G.; Wilson, S.E. Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest. Ophthalmol. Vis. Sci. 2001, 42, 2795–2803.
[30]
Bourcier, T.; Sauer, A.; Letscher-Bru, V.; Candolfi, E. Fungal keratitis. J. Fr. Ophtalmol. 2011, 34, 563–567, doi:10.1016/j.jfo.2011.03.001.
[31]
Chee, S.P.; Jap, A. Immune ring formation associated with cytomegalovirus endotheliitis. Am. J. Ophthalmol. 2011, 152, 449–453, doi:10.1016/j.ajo.2011.03.003.
García-Pérez, B.E.; Villagomez-Palatto, D.A.; Casta？eda-Sánchez, J.I.; Coral-vázquez, R.M.; Ramírez-Sánchez, I.; Ordo？ez-Razo, R.M.; Luna-Herrera, J. Innate response of human endothelial cells infected with mycobacteria. Immunobiology 2011, 216, 925–935.
[36]
Sanguinetti, M.; Ardito, F.; Fiscarelli, E.; La Sorda, M.; D'Argenio, P.; Ricciotti, G.; Fadda, G. Fatal pulmonary infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis. J. Clin. Microbiol. 2001, 39, 816–819, doi:10.1128/JCM.39.2.816-819.2001.
[37]
Ham, H.; Sreelatha, A.; Orth, K. Manipulation of host membranes by bacterial effectors. Nat. Rev. Microbiol. 2011, 9, 635–646, doi:10.1038/nrmicro2602.
[38]
Dickinson, J.M.; Mitchison, D.A. Experimental models to explain the high sterilizing activity of rifampin in the chemotherapy of tuberculosis. Am. Rev. Respir. Dis. 1981, 123, 367–371.
[39]
Arriaga, A.K.; Orozco, E.H.; Aguilar, L.D.; Rook, G.A. Hernández Pando R. Immunological and pathological comparative analysis between experimental latent tuberculous infection and progressive pulmonary tuberculosis. Clin. Exp. Immunol. 2002, 128, 229–237, doi:10.1046/j.1365-2249.2002.01832.x.
Yuan, X.; Hua, X.; Wilhelmus, K.R. The corneal expression of antimicrobial peptides during experimental fungal keratitis. Curr. Eye Res. 2010, 35, 872–879.
[44]
Gallo, R.L.; Murakami, M.; Ohtake, T.; Zaiou, M. Biology and clinical relevance of naturally occurring antimicrobial peptides. J. Allergy Clin. Immunol. 2002, 110, 823–831.
[45]
Rivas-Santiago, B.; Contreras, J.C.; Sada, E.; Hernandez-Pando, R. The potential role of lung epithelial cells and beta-defensins in experimental latent tuberculosis. Scand. J. Immunol. 2008, 67, 448–452.
[46]
Rivas-Santiago, B.; Sada, E.; Tsutsumi, V.; Aguilar-Leon, D.; Contreras, J.L.; Hernandez-Pando, R. Beta-Defensin gene expression during the course of experimental tuberculosis infection. J. Infect. Dis. 2006, 194, 697–703.
[47]
Medzhitov, R. 2009. Damage control in host-pathogen interactions. Proc. Natl. Acad. Sci. USA 2009, 106, 15525–15526, doi:10.1073/pnas.0908451106.
Leal, I.S.; Smedegard, B.; Andersen, P.; Appelberg, R. Interleukin-6 and interleukin-12 participate in induction of a type 1 protective T-cell response during vaccination with a tuberculosis subunit vaccine. Infect. Immun. 1999, 67, 5747–5754.
[50]
Dutta, R.K.; Kathania, M.; Raje, M.; Majumdar, S. IL-6 inhibits IFN-γ induced autophagy in Mycobacterium tuberculosis H37Rv infected macrophages. Int. J. Biochem. Cell. Biol. 2012, 44, 942–954.
[51]
Tamai, R.; Sugamata, M.; Kiyoura, Y. Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis. Microb. Pathog. 2011, 51, 250–254, doi:10.1016/j.micpath.2011.06.009.
[52]
Li, J.; Raghunath, M.; Tan, D.; Lareu, R.R.; Chen, Z.C.; Beuerman, R.W. Defensins HNP1 and HBD2 stimulation of wound-associated responses in human conjunctival fibroblasts. Invest. Ophthalmol. Vis. Sci. 2006, 47, 3811–3819.
[53]
Li, J.; Zhu, H.Y.; Beuerman, R.W. Stimulation of specific cytokines in human conjunctival epithelial cells by defensins HNP1, HBD2 and HBD3. Invest. Ophthalmol. Vis. Sci. 2009, 50, 644–653.
[54]
Larrick, J.; Hirata, M.; Balint, R.; Lee, J.; Zhong, J.; Wright, S.C. Human CAP 18: a novel antimicrobial lipopolysaccharide-binding protein. Infect. Immun. 1995, 63, 1291–1297.
[55]
García-Pérez, B.E.; Hernández-González, J.C.; García-Nieto, S.; Luna-Herrera, J. Internalization of a non-pathogenic mycobacteria by macropinocytosis in human alveolar epithelial A549 cells. Microb. Pathog. 2008, 45, 1–6, doi:10.1016/j.micpath.2008.01.009.
[56]
Sonawane, A.; Santos, J.C.; Mishra, B.B.; Jena, P.; Progida, C.; Sorensen, O.E.; Gallo, R.; Appelberg, R.; Griffiths, G. Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages. Cell. Microbiol. 2011, 13, 1601–6017, doi:10.1111/j.1462-5822.2011.01644.x.
[57]
Chaurasia, S.S.; Kaur, H.; de Medeiros, F.W.; Smith, S.D.; Wilson, S.E. Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury. Exp. Eye Res. 2009, 89, 133–139, doi:10.1016/j.exer.2009.02.022.
[58]
Liu, J.; Song, G.; Wang, Z.; Huang, B.; Gao, Q.; Liu, B.; Xu, Y.; Liang, X.; Ma, P.; Gao, N.; Ge, J. Establishment of a corneal epithelial cell line spontaneously derived from human limbal cells. Exp. Eye Res. 2007, 84, 599–609, doi:10.1016/j.exer.2006.11.014.
