Experimental sporotrichosis in a cyclophosphamide-induced immunosuppressed mice model

Medical Mycology, 2018, 56, 711–722 doi: 10.1093/mmy/myx098 Advance Access Publication Date: 26 October 2017 Original Article Original Article Experimental sporotrichosis in a cyclophosphamide-induced immunosuppressed mice model Francine Alessandra Manente# , Camila Quinello# , Lucas Souza Ferreira, Cleverton Roberto de Andrade, Juliana Aparecida Jellmayer, Deivys Leandro Portuondo, Alexander Batista-Duharte and Iracilda Zeppone Carlos∗ School of Pharmaceutical Sciences, São Paulo State University – UNESP, Araraquara, SP, Brazil ∗ To whom correspondence should be addressed. Iracilda Zeppone Carlos, PhD, São Paulo State University - UNESP, School of Pharmaceutical Sciences, Department of Clinical Analysis, Rodovia Araraquara-Jaú - Km 1 Postal Code: 14800-903 Araraquara, SP, Brazil. Tel: +55 16 3301 5712; Fax: +55 16 3322 0073; E-mail: (I.Z. Carlos). # They are considered first authors. Received 12 April 2017; Revised 23 June 2017; Accepted 6 September 2017; Editorial Decision 3 July 2017 Abstract This report describes a model of host resistance for Sporothrix schenckii, an opportunistic fungi in immunosuppressed mice with cyclophosphamide (CY) to be used in studies of immunotoxicology and immunopharmacology. Two doses of CY were administered intraperitoneally: 200 mg/kg and a booster of 150 mg/kg at 9-day intervals. Three days after the first dose of CY the animals were infected subcutaneously with 1.8 × 108 yeast/ml (S. schenckii ATCC 16345). At 7 and 14 days post-infection, the animals were euthanized and analyzed the fungal load by unit forming colony count in the spleen and popliteal lymph nodes. The relative weight of thymus and spleen, splenic index, the frequency of T and B cells in spleen by flow cytometry, the hind paw inflammation index and cytokine (interleukin [IL]-17, IL-10, and interferon [IFN]-γ ) profile were measured. Histopathological studies of the spleen and the hind paw were also assessed. The immunosuppression status was confirmed at the evaluated days by reduction of relative weight of thymus, reduction of the splenic white pulp, the population of B and T lymphocytes, and the cytokine profile in the treated mice with CY in comparison with nontreated groups, associated to higher fungal load in hind paw and spleen in the infected mice. The described model reveals an increasing in susceptibility to infection and severity when associated with immunosuppression. This model can serve as a reference for studies of S. schenckii host resistance in pharmaceutical and toxicological studies. Key words: Sporothrix schenckii, host resistance models, immunosuppression, cyclophosphamide.  C The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: 711 712 Introduction Cyclophosphamide (CY) is an alkylating agent that can inhibit both the humoral and cellular immune responses. It is widely used as cytotoxic drug in the anticancer therapy and as immunosuppressive drug in autoimmune diseases and organ transplants, including prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation.19–22 CY and Methotrexate (MTX) are the more frequently reported antineoplastic drugs associated to sporotrichosis. They are administrated in combined therapy joint to other immunosuppressive drug.23–27 In 1981, the susceptibility to sporotrichosis in a murine model of cyclophosphamide immunosuppression was described.28 However, no further description of S. schenckii as host resistance model in immunosuppressed mice, using quantitative immunological biomarkers were made. This study aimed to develop a murine model of host resistance to S. schenckii sensu stricto in immunosuppressed mice with CY as a tool for future studies of immunotoxicity and immunopharmacology in sporotrichosis. Methods Animals Male Balb/c mice, 5–7 week old at the time of inoculation, were purchased from “Centro Multidisciplinar para Investigação Biológica na Área da Ciência de Animais de Laboratório” (CEMIB), Universidade de Campinas (UNICAMP), São Paulo, Brazil. Animals were housed in individually ventilated cages in an ambient with controlled temperature and 12-h light/dark cycles. Water and food were offered ad libitum. All procedures were approved by the Institutional Ethics Committee (Protocol CEP/FCF/CAR no. 04/2014) and were in accordance with the National Institutes of Health Animal Care Guidelines. Microorganism and culture conditions R S schenckii (ATCC 16345TM ) isolated from a case of human lung infection (Baltimore, MD, USA), was kindly provided by Department of Microbiology, Materials laboratory references the Oswaldo Cruz Foundation, National Institute of Quality Control in Health, Rio de Janeiro, Brazil. Currently this isolated sustained in the Department of Clinical Analysis, Araraquara’s School of Pharmaceutical Sciences, Universidade Estadual Paulista-UNESP, Júlio Mesquita Filho, in the filamentous phase amid MycoselTM at room temperature (±25◦ C). The yeast form is obtained in BHI (Brain Heart Infusion, Difco) at 37◦ C under constant stirring of 150 rpm / min for 5 days. Despite the existence of sophisticated molecular and genetic techniques to identify defects in resistance pathways at the molecular and message level, the host resistance assays using opportunist microorganisms remain the gold standard against which changes at the molecular or cellular level of immune function can be judged. This is due to resistance to infection, regardless of the actual pathogen, involves multiple pathways of effectors’ function to neutralize or eliminate pathogens.1 Isolated or multiple tests are even insufficient to evaluate the consequences of the immunosuppression for the risk of specific infections in several cases2 . Sporotrichosis is a chronic fungal infection caused by different species of Sporothrix that are classified into five clades based on gene sequences of chitin synthase, β-tubulin and calmodulin: Sporothrix brasiliensis (Clade I); Sporothrix schenckii (Clade II); Sporothrix globosa (Clade III) Sporothrix mexicana (Clade IV) and Sporothrix pallida (formerly S. albicans) (Clade V).3 Sporotrichosis has been described on five continents, with a higher prevalence in tropical and subtropical regions.4,5 Infection generally occurs following traumatic inoculation with soil, plants, and organic matter contaminated with the fungus causing cutaneous and subcutaneous infection, leading to plaque formation as warts and lesions papules. Both manifestations can progress to ulcerative lesions. The main clinical manifestations of sporotrichosis are the lymphocutaneous forms, but the number of systemic and visceral cases has increased, particularly in immunodeficient patients.6 Zoonotic transmission has been increasing significantly, with a recent report of an epidemic outbreak with zoonotic transmission by infecte (...truncated)