Galectin-3 plays an important role in protection against disseminated candidiasis

Medical Mycology August 2013, 51, 641–651 Galectin-3 plays an important role in protection against disseminated candidiasis JENNIFER R. LINDEN*, MONIQUE E. DE PAEPE†, SONIA S. LAFORCE-NESBITT‡ & JOSEPH M. BLISS*‡ *Graduate Program in Pathobiology, Brown University, Providence, RI, and Departments of †Pathology and ‡Pediatrics,Women & Infants Hospital of Rhode Island, Warren Alpert Medical School of Brown University, Providence, RI, USA Keywords Galectin-3, Candida albicans, Candida parapsilosis, candidiasis, neonate Introduction Candida species are the fourth most common cause of nosocomial bloodstream infections and are the leading cause of invasive fungal disease [1,2]. Historically, Candida albicans has been the leading cause of invasive fungal infections; however, the frequency of infections caused by non-albicans species has dramatically increased [3–5]. Numerous studies have reported that infections caused by C. parapsilosis account for 15–67% of invasive candidiasis in premature infants, and in some centers this species surpasses C. albicans as the predominant cause of neonatal candidiasis [6]. Interestingly, C. parapsilosis is often associated with lower mortality rates in the human host Received 1 October 2012; Received in final revised form 4 December 2012; Accepted 7 January 2013 Correspondence: Joseph M. Bliss, Department of Pediatrics, Women & Infants Hospital of Rhode Island, Warren Alpert Medical School of Brown University, Providence, RI 02905, USA. Tel.: ⫹ 1 401 274 1100; Fax: ⫹ 1 401 453 7571. E-mail: © 2013 ISHAM and is significantly less virulent than C. albicans in mouse models of infections [7–12]. Nevertheless, systemic infections caused by both species are associated with significant morbidity and mortality rates, even with antifungal treatment [2,4,13]. Understanding how the host defends against these infections will further our knowledge of patient susceptibilities and aid in the development of novel therapeutics. To defend against pathogenic fungi, the host’s immune system must respond to the invading pathogens through recognition of specific fungal pathogen associated molecular patterns (PAMPs). These PAMPs are recognized by pathogen recognition receptors (PRRs) which are found on a wide array of cells [14,15]. PRRs include C-type lectin receptors, toll-like receptors (TLRs), integrins, and scavenger receptors. Among C-type lectin receptors, dectin-1 recognizes β-(1,3)-glucan, dectin-2 recognizes high mannose structures, and the mannose receptor recognizes N-linked mannan. Among TLRs, TLR2 recognizes β-(1–2)-mannose structures and phospholipid mannan, TLR4 recognizes O-linked mannan, and TLR6 DOI: 10.3109/13693786.2013.770607 Recent in vitro studies have implicated galectin-3 as an important receptor in host recognition and response to specific Candida species; however, its role in protection against disseminated candidiasis in vivo has not been evaluated. This study investigated the importance of galectin-3 in host defense against systemic infection with the highly virulent species Candida albicans, and the less virulent species, C. parapsilosis. Mice deficient in galectin-3 (gal3⫺/⫺) were more susceptible to infection than wild-type (WT) mice. When infected with C. albicans, gal3⫺/⫺ mice died significantly faster and exhibited a trend towards increased fungal burden and increased abscess formation in infected brains compared to WT mice. When infected with C. parapsilosis, gal3⫺/⫺ mice had significantly higher renal fungal burdens and abscess formation compared to WT mice. To evaluate whether galectin-3 may contribute to susceptibility to candidiasis in human infants, galectin-3 levels in sera of newborn infants, a patient population uniquely susceptible to infections with both C. albicans and C. parapsilosis, were compared to serum galectin-3 levels of adults. Galectin-3 levels were significantly lower in newborn infant sera compared to adult sera. These data indicate that galectin-3 plays an important role in a murine model of disseminated candidiasis and suggest a potential mechanism of neonatal susceptibility to these infections. 642 Linden et al. Materials and methods Growth, maintenance, and preparation of organisms C. albicans strain SC5314 and a C. parapsilosis clinical isolate, 15-72391-101 [39], referred to here as JMB81, were used throughout this study. Strains were maintained on YPD plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar). To prepare strains for injection, overnight cultures were grown for 16 h at 37°C with vigorous agitation in YPD broth. Cells were harvested and washed six times by centrifugation in sterile saline. C. albicans was adjusted to 5 ⫻ 106 cells/ml and C. parapsilosis was adjusted to 5 ⫻ 108 cells/ml in sterile saline. Prepared cell suspensions were used immediately to infect mice. Suspension concentrations were confirmed by serial dilution onto YPD plates incubated at 37°C for 48 h. Murine model of disseminated candidiasis Following review and approval by the Institutional Animal Welfare Committee, 4–8-week-old female gal3⫺/⫺ mice (B6.Cg-Lgals3 tm Poi/J) and their WT counterparts (C57BL/6J) were obtained from Jackson Laboratories. Mice were infected with 1 ⫻ 105 CFU of C. albicans strain SC5314 or 1 ⫻ 108 CFU of C. parapsilosis strain JMB81 by injecting 200 μl of the cell suspension prepared above via tail vein. Survival was monitored up to 21 days post infection. All procedures involving animals conformed to the ILAR Guide for the Care and Use of Laboratory Animals (2011 edition) of the Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. Fungal burden analysis of harvested organs To determine the fungal burdens of mice infected with C. albicans, mice were euthanized at 3 days post infection or when moribund and organs were harvested for CFU counts. To determine fungal burdens of mice infected with C. parapsilosis, mice were euthanized 3, 7 and 21 days post infection. Harvested organs were weighed, placed in 1 ml of sterile saline, and homogenized using a FastPrep®-24 Tissue and Cell Homogenizer (MP Biomedicals) with Lysing Matrix D. Homogenized organs were serially diluted and plated onto YPD plates containing streptomycin (100 μg/ml) and ampicillin (50 μg/ml). Inoculated plates were incubated at 37°C for 48–72 h before enumerating CFUs. Fungal burden was expressed as the number of CFU/ml/gram of harvested organ. Histological examinations of harvested organs Brains and kidneys of infected mice were fixed in 10% formalin at time of death. Fixed organs were sectioned and stained with Grocott’s methenamine silver (GMS) or hematoxylin and eosin (H&E). For H&E stained slides, the number of brain and renal abscesses were counted during microscopic examination at high magnification and expressed as the number of abscesses per tissue section. Galectin-3 ELISA Following review and approval by the Institutional Review Board, sera from periphera (...truncated)