Research letter

Photodynamic therapy efficiently controls dermatophytosis caused by Trichophyton rubrum in a murine model DOI: 10.1111/bjd.13494 DEAR EDITOR, Dermatophytes are filamentous fungi that use keratin to colonize the host, causing superficial infections called dermatophytoses. Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum spp. and Epidermophyton spp. are important aetiological agents worldwide.1 A few drugs, including azoles (itraconazole), allylamines (terbinafine) and hydroxypiridones [ciclopirox olamine (CPX)], are available for the treatment of dermatophytoses,1,2 but the widespread use of antifungals has been linked to the emergence of resistant strains of T. rubrum.2 This problem may jeopardize treatment and thereby justifies the search for new therapeutic strategies. Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer and a nonthermal light source to induce a reaction that results in cell death.3 In the presence of molecular oxygen, aPDT triggers the production of reactive oxygen (ROS) and reactive nitrogen species,4 which have short lifetimes and limited diffusion through the tissue, explaining the localized damage induced by aPDT.3,4 Previously, we have shown that T. rubrum is susceptible to photodynamic inhibition using toluidine blue (TBO) and light-emitting diode (LED) at 630 nm;4 however, there is a lack of studies regarding its efficacy in vivo.5 Therefore, the aim of this study was to evaluate the effects of aPDT, using

TBO and LED, in controlling T. rubrum infection in a murine model of dermatophytosis. In this study, C57BL/6 mice were infected with 1 9 106 conidia per animal of T. rubrum American Type Culture Collection 28189 and treated with aPDT daily [using TBO 0
2% in a gel formulation (Sigma-Aldrich, St Louis, MO, U.S.A.) and 630-nm LED (Fisioled; MMoptics, S~ao Paulo, Brazil) at a dose of 42 J cm2] or CPX [10 mg g 1 (dose of 0
65 mg 1 per mouse); Medley, S~ao Paulo, Brazil] for 48 h, over a period of 7 days (Comiss~ao de Etica no Uso de Animals/Universidade Federal de Minas Gerais ethics protocol approval no. 040/ 2011).6 Histopathology of the skin, fungal burden in potato agar, and myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activity were evaluated.6 The influence of aPDT on the viability of intraperitoneal macrophages, intracellular formation of hyphae and oxidative burst (ROS) was determined.4,6 For this purpose, TBO (10 lg mL 1) and LED (48 J cm2) were used. The groups of mice and macrophages used in this work are described in Table 1. Histopathological analyses showed that aPDT reduced the signs of dermatitis and recovered tissue architecture (Fig. 1c, g,i) compared with the infected and nontreated group (Fig. 1b,f,i). Interestingly, fungal cells were found mainly in hair follicles, corroborating the keratinophilic aspect of T. rubrum (Fig. 1f–h). Indeed, aPDT significantly reduced the fungal burden by 87% compared with the untreated group (P < 0
01) and by 64% compared with the CPX group (P < 0
05) (Fig. 1d,f–h,j). LED or TBO alone did not reduce the fungal burden (Fig. 1j). In addition, aPDT significantly

Table 1 Groups and conditions for antimicrobial photodynamic therapy in vivo and in vitro Group NI NT LED TBO aPDT CPX Ma NT LED TBO aPDT

Treatment type In vivo Uninfected mice Mice infected with T. rubrum and without any treatment Mice infected with T. rubrum and treated daily with LED light dose of 42 J cm2 Mice infected with T. rubrum and treated daily with TBO 0
2% gel for 10 min under light protection Mice treated daily with PDT (LED dose of 42 J cm2 and TBO 0
2% gel) Mice treated topically with CPX cream (0
65 mg 1 mice every 48 h) In vitro Uninfected (macrophages only) Macrophages challenged with T. rubrum conidia and without any treatment Macrophages challenged with T. rubrum conidia and treated with an LED dose of 48 J cm2 Macrophages challenged with T. rubrum and treated with TBO 10 lg mL 1 Macrophages challenged with T. rubrum and treated with PDT (LED dose of 48 J cm2 and TBO 10 lg mL 1)

T. rubrum, Trichophyton rubrum; LED, light-emitting diode; TBO, toluidine blue; PDT, photodynamic therapy; CPX, ciclopirox olamine.

© 2014 British Association of Dermatologists

British Journal of Dermatology (2015) 172, pp801–804

801

802 Research letter

(a)

(e)

(b)

(f)

(i)

(j)

(k) (c)

(g)

(d)

(h)

(l)

Fig 1. Histopathological analysis, and fungal burden, myeloperoxidase (MPO) and N-acetylglucosamine (NAG) assays of the skin of mice. Histopathological analysis of the skin of uninfected mice stained with (a) haematoxylin and eosin (H&E) and (e) Gomori–Grocott (G & G); untreated group stained with (b) H&E and (f) G & G; antimicrobial photodynamic therapy (aPDT) group stained with (c) H&E and (g) G & G; and ciclopirox olamine (CPX) group stained with (d) H&E (h) and G & G. (a–h) Images are representative of one animal per group. Scale bar on images on the left = 400 lm; scale bar on images on the right = 100 lm. Unfilled star, dermatitis; triangle, fibrosis; asterisks, hyperaemia; arrow, hyperkeratosis; filled triangle, fungal cell within of the hair follicle. (i) Semiquantitative analyses of skin damage presented by NI, untreated, aPDT and CPX groups. (j) Fungal burden [colony forming units (CFU) g 1 of skin]. (k) MPO activity. (l) NAG activity. (i–l) Results are expressed as mean  SEM (n = 6 mice per group). *P < 0
05 compared with NI group; ***P < 0
0001 compared with NI group; #P < 0
05 compared with the NT group; ##P < 0
001 compared with the NT group. See Table 1 for the definitions of each group.

(P < 0
05) reduced the activity of MPO but did not influence that of NAG (Fig. 1k,l). The phagocytosis assay showed that aPDT preserved the viability of macrophages and reduced the viability of T. rubrum. The group that underwent aPDT presented cell viability similar to that of the macrophage-only group and higher than that of the untreated group (Fig. 2a), and a lower number of hyphae when compared with the untreated group (Fig. 2b). The generation of

(a)

(b)

ROS was significantly (P < 0
05) higher in the aPDT group compared with the macrophage-only and untreated groups (Fig. 2c). The study of PDT as a treatment is recent, and data regarding its efficacy in animal models are scarce. To date, a small number of studies have shown the effectiveness of aPDT in treating bacterial infection;7 however, to our knowledge, no studies have used animal models to demonstrate that aPDT

(c)

Fig 2. (a) Viability of macrophages challenged with Trichophyton rubrum conidia and treated with antimicrobial photodynamic therapy (aPDT). (b) Intracellular hyphae formation within macrophages. (c) Production of ROS production. The results shown in (a–c) are expressed as mean  SEM. *P < 0
05 compared with Ma; #P < 0
05 compared with the untreated group. See Table 1 for the definitions of each group. OD, optical density; DCF-DA, dichloro-dihydro-fluorescein diacetate. British Journal of Dermatology (2015) 172, pp801–804

© 2014 British Association of Dermatologists

Research letter

using LED and TBO can be efficient in controlling dermatophytosis caused by T. rubrum. LED emitting at 630 nm is resonant with TBO and was chosen because it penetrates deeply into the skin, reaching both stratum corneum and hair follicles.3 Treatment with LED or TBO alone did not alter the fungal burden, confirming that the combination of both was crucial for the effect of aPDT. To our knowledge, this is the first study to demonstrate that aPDT can be more efficient than an antifungal drug in controlling T. rubrum infection, encouraging future studies with aPDT against other dermatophytes species. In this study we worked with a formulation of CPX available for human use. The schedule of CPX application was chosen based on the combination of CPX with aPDT. We expected to be able to reduce the dose of CPX and to see a reduction in adverse effects; however, this combination did not improve the condition of mice compared with each treatment alone (data not shown). Clinical investigation should be considered given the unspecific mechanism of death of the microorganism triggered by aPDT, which was unlike that of conventional antifungal drugs.8 Moreover, treatment with CPX can cause adverse effects such as irritation, burning, itch and pain.9 The TBO gel formulation proved to be an efficient delivery system for topical application of the photosensitizer as it minimizes the risk of systemic absorption, reducing the possibility of side-effects. Furthermore, aPDT reduced the fungal burden and signs of disease without causing tissue damage, probably because it selectively killed T. rubrum rather than host cells. The small size of conidia (with less availability of antioxidant molecules and a higher concentration of ROS) compared with mammalian cells may explain the lower resistance of fungal cells to the oxidative burst generated by aPDT. As the murine model of dermatophytosis by T. rubrum previously described by our group showed higher fungal recovery at 7 days postinfection,6 we therefore began the treatment at this time. The increased levels of MPO (except for the aPDT group) and NAG indicate the recruitment of neutrophils and macrophages to the site of infection, respectively, reinforcing the importance of these cells in controlling fungal burden.6,10 Although the role of PDT-activated neutrophils is well established,11 the reduction of neutrophils in the skin seen after aPDT was related to control of the infection. Interestingly, NAG activity remained elevated after aPDT, suggesting that macrophages were involved in the control of fungal burden (by engulfing and killing the fungus) and in the resolution of infection (by the phagocytosis of cellular debris). Indeed, the death of T. rubrum triggered by aPDT probably augments the exposition of antigens and, consequently, increases its recognition by macrophages.4 This recruitment corroborates the therapeutic use of red light, which improves the immune system, and increases blood flow and wound healing.12 The phagocytosis assay showed that aPDT caused selective damage to T. rubrum cells instead of macrophages. The fungal cells inside macrophages presented a reduced ability to produce hyphae after aPDT, preventing the death of the host cells. This is an important © 2014 British Association of Dermatologists

803

finding, as different studies have reported that the formation of hyphae is the major cause of macrophage death during infection by filamentous fungi.6,10 Finally, the higher levels of ROS seen after aPDT probably impaired the germination of conidia and fungal growth.6,10 In conclusion, in comparison with an untreated group, we have shown that aPDT using LED and TBO reduced the fungal burden in mice infected with T. rubrum. The fungicidal effect was related to the production of ROS, which is important in reducing fungal viability and preventing injuries caused by the fungus without damaging host tissue.

Acknowledgments We thank Dr C.M. Queiroz-Junior for assistance with the histopathological procedures and for constructive comments, and Gilv^ania Ferreira Silva Santos for technical support. 1

Departments of Microbiology, Pharmaceutical Products and 3 Mechanical Engineering, Instituto de Ci^encias Biologicas, Universidade Federal de Minas Gerais, Av. Presidente Ant^onio Carlos, 6627, Pampulha 31270-901, Belo Horizonte, Minas Gerais, Brazil. Correspondence: Daniel A. Santos. E-mail: [email protected] 2

L.M. BALTAZAR1 S.M.C. WERNECK1 H.C.S. CARNEIRO1 L.F. GOUVEIA1 T.P. DE PAULA1 R.M.D. BYRRO2 A . S . C U N H A J U N I O R 2 B.M. SOARES3 M.V.L. FERREIRA3 D.G. SOUZA1 M. PINOTTI3 P.S. CISALPINO1 D.A. SANTOS1

References 1 Weitzman I, Summerbell RC. The dermatophytes. Clin Microbiol Rev 1995; 8:240–59. 2 Gupta AK, Cooper EA. Update in antifungal therapy of dermatophytosis. Mycopathologia 2008; 166:353–67. 3 Calzavara-Pinton P, Rossi MT, Sala R et al. Photodynamic antifungal chemotherapy. Photochem Photobiol 2012; 88:512–22. 4 Baltazar Lde M, Soares BM, Carneiro HC et al. Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death. J Antimicrob Chemother 2013; 68:354–61. 5 Smijs TG, Pavel S. The susceptibility of dermatophytes to photodynamic treatment with special focus on Trichophyton rubrum. Photochem Photobiol 2011; 87:2–13. 6 Baltazar Lde M, Santos PC, Paula TP et al. IFN-c impairs Trichophyton rubrum proliferation in a model murine of dermatophytosis through the production of IL-1b and reactive oxygen species. Med Mycol 2014; 52:293–302. 7 Lin J, Bi LJ, Zhang ZG et al. Toluidine blue-mediated photodynamic therapy of oral wound infections in rats. Lasers Med Sci 2010; 25:233–8. 8 Gupta AK. Ciclopirox: an overview. Int J Dermatol 2001; 40:305–10. 9 Subissi A, Monti D, Togni G et al. Ciclopirox: recent nonclinical and clinical data relevant to its use as a topical antimycotic agent. Drugs 2010; 70:2133–52. British Journal of Dermatology (2015) 172, pp801–804

804 Research letter 10 Bonnett CR, Cornish EJ, Harmsen AG et al. Early neutrophil recruitment an aggregation in the murine lung inhibit germination of Aspergillus fumigatus conidia. Infect Immun 2006; 74:6528–39. 11 Tanaka M, Mroz P, Dai T et al. Photodynamic therapy can induce a protective innate immune response against murine bacterial arthritis via neutrophil accumulation. PLoS One 2012; 7:e39823. 12 Okuni I. Phototherapy in rehabilitation medicine. Masui 2012; 61:700–5 (in Japanese).

British Journal of Dermatology (2015) 172, pp801–804

Funding sources: Coordenacß~ao de Aperfeicßoamento de Pessoal de Nıvel Superior (CAPES) and Fundacß~ao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG). Conflicts of interest: none declared.

© 2014 British Association of Dermatologists