Eur J Clin Microbiol Infect Dis (2012) 31:1271–1277 DOI 10.1007/s10096-011-1439-z
RESEARCH ARTICLE
Activation pattern of neutrophils from blood of elderly individuals with Candida-related denture stomatitis T. H. Gasparoto & C. E. de Oliveira & N. A. Vieira & V. C. Porto & F. Q. Cunha & G. P. Garlet & A. P. Campanelli & V. S. Lara
Received: 4 March 2011 / Accepted: 23 September 2011 / Published online: 26 November 2011 # Springer-Verlag 2011
Abstract We have identified impaired neutrophils in elderly individuals which could be involved with Candida-related denture stomatitis (DS), an oral infection predominantly caused by Candida albicans, affecting especially elderly individuals using dental prosthesis. However, specific mechanisms performed by neutrophil contributing to the T. H. Gasparoto : G. P. Garlet : A. P. Campanelli Department of Biological Sciences, University of São Paulo (USP), Bauru, SP, Brazil C. E. de Oliveira : V. S. Lara Department of Stomatology (Oral Pathology), University of São Paulo (USP), Bauru, SP, Brazil N. A. Vieira Clinical Analysis Laboratory of the Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo (USP), Bauru, SP, Brazil V. C. Porto Department of Prosthodontics - Bauru School of Dentistry, University of São Paulo (USP), Bauru, SP, Brazil F. Q. Cunha Department of Pharmacology, School of Medicine of Ribeirão Preto – University of São Paulo (USP), Ribeirão Preto, SP, Brazil T. H. Gasparoto (*) Department of Biological Sciences, Bauru School of Dentistry (FOB/USP), 9-75 Octávio Pinheiro Brisola, 17012-901 Bauru, SP, Brazil e-mail:
[email protected] T. H. Gasparoto e-mail:
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susceptibility of the elderly to DS are not fully understood. This study evaluated activation features of blood neutrophils from elderly and young individuals with DS. Blood neutrophils cultured with C. albicans from elderly subjects secreted decreased levels of CXCL8. However, C. albicans challenged-neutrophils from DS patients produced high IL4 and IL-10, and low GM-CSF levels, regardless of age. Additional elastase activity of neutrophils from both elderly groups was detected after incubation with C. albicans, but only neutrophils from elderly DS demonstrated high myeloperoxidase activity. Therefore, DS patients have affected neutrophils, and the advance of age intensifies these damages. In sumamry, individuals with Candidarelated denture stomatitis presented variation in the neutrophil phenotype and activation. Such alterations were more intense in neutrophils from infected elderly individuals.
Introduction Neutrophils are responsible for early cytokine and chemokine secretion and the release of antimicrobial factors, such as reactive oxygen and nitrogen species in response to pathogens, including Candida species [1–6]. Candidarelated denture stomatitis (DS) is an inflammatory condition frequently observed in elderly individuals caused primarily by Candida albicans, an ubiquitous fungal organism that is part of the normal oral microflora in up to 80% of healthy individuals [7]. The nature of the protective responses against C. albicans is relatively well established in the current literature, with the neutrophils implicated as an important leukocyte subset in the control of this fungus [1, 2, 6]. Although PMNs are shown to be active in the innate immune response at these primary barriers to systemic invasion, their influence in the differ-
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ences between the local immunity of the oral mucosa have been demonstrated [7–9]. Products of neutrophils activation are important in the attraction and activation of leukocytes involved in the development of Th1-type protective response against candidiasis [3, 8, 9]. In fact, neutrophils have been shown to influence antifungal Th-cell development, as evidenced by the inability of neutropenic mice to perform a protective Th1 response to C. albicans infection [3, 8, 9]. Among the factors described significantly to affect neutrophil function, ageing is characterized by drastic changes of the immune system, usually known as immunosenescence [10, 11]. In fact, ageing is associated with the deterioration of the immune cells facilitating the establishment and exacerbation of infections [12]. Specifically changes in neutrophils’ function and phenotype are described in elderly subjects [10, 11]. Accordingly, we have previously demonstrated that DS in elderly individuals is correlated to impaired function of neutrophils against C. albicans [7]. Even though the importance of neutrophils against candidosis is well known, the majority of these studies aimed at disseminated, but not localized diseases [13]. Besides, there are few studies evaluating blood neutrophils from individuals with DS. In the present work, we evaluated the production of cytokines and chemokines involved with protection against C. albicans as well as elastase (ELA) and myeloperoxidase (MPO) activities of blood neutrophils from elderly and young individuals with DS responding to Candida challenge. Our objective was to determine possible features in neutrophils which could be collaborating in the establishment and persistence of DS in the elderly.
Materials and methods Subjects The patients were drawn from Bauru School of Dentistry, Prosthodontics Clinic, University of Sao Paulo (USP), after obtaining the informed consent from each individual in compliance with resolution 196/96 of the National Council of Health and University of São Paulo (Bauru, São Paulo, Brazil) committee guidelines. The inclusion criteria comprised aged subjects presenting DS, which had been confirmed by microbiological diagnosis (elderly DS, n= 15) or healthy subjects (elderly control, n=15), ranging from 60 to 85 years old (mean age 69.5±1.6 years old for elderly patients and 67.9±1.2 years old in control groups). Additionally, individuals ranging from 25 to 50 years old presenting DS (young DS, n=15, mean age 46.8±1.5 years) or not (young control, n=15, mean age 38.9±3.1 years)
Eur J Clin Microbiol Infect Dis (2012) 31:1271–1277
were assessed. Age limits were determined based on a previous study [14]. Complete medical and dental histories were recorded. None of the individuals had been treated previously in a sample collection regarding DS. The exclusion criteria were patients suffering from the following conditions: diabetes mellitus, alcoholism, tobacco use, periodontal diseases or other oral pathologies, gingival bleeding, cancer and symptoms that could indicate cancer such as sudden changes in weight and appetite, immune, endocrine or hematologic alterations and the use of xerostomic, antifungal or antibiotic medications. Participants who presented any other oral infectious diseases and active dental abscesses were excluded from the study. Identification of Candida-related denture stomatitis DS was clinically diagnosed as pinpoint hyperaemia, diffuse or granular, as previously reported [15]. Microbiologic diagnosis was achieved as earlier described [16]. Samples were collected from hard palate in an erythematous area with a sterile swab. All samples were grown on Sabouraud dextrose broth (Difco, Becton Dickinson) with 1% chloramphenicol (Sigma-Aldrich) at room temperature. This procedure was completed in order to avoid false-negativity for the presence of Candida. Blood neutrophils purification All blood samples were collected between 7:00 and 9:00 a. m. Heparinized whole blood (10 mL) was obtained by venipuncture, and neutrophils were purified. Briefly, 5 mL of Histopaque 1119 (Sigma-Aldrich Brazil Ltd., São Paulo, Brazil) were poured into a 15-mL round bottom tube and overlaid with 3 mL of Histopaque 1083 (Sigma-Aldrich Brazil Ltd.), and 6 mL of whole blood were layered over the gradients. Samples were centrifuged (460 X g) for 28 min at 25°C. The second band, which contained the neutrophils, was aspirated and washed twice with cold RPMI 1640 (GIBCO). Neutrophils were identified through staining with Türk’s solution. Neutrophil viability was analyzed by either positivity for annexin V-FITC and/or propidium iodide (Aposcreen Annexin V-FITC, R&D Systems, Minneapolis, USA). More than 99% of the neutrophils were viable immediately before the culture assays. We assured purity of the samples through flow cytometry analysis considering CXCR1+ cells as neutrophils after gated following SSC X FSC parameters. Candida albicans ATCC 10231 C. albicans ATCC 10231 strains were obtained from the stock culture collection of the Department of Immunol-
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ogy and Microbiology, Bauru School of Dentistry, University of São Paulo (USP). Yeast cells were grown on Sabouraud dextrose agar and maintained at 28°C. Before assays, blastoconidia were grown in Sabouraud dextrose broth for 48 h at room temperature. At the moment of the culture assay, these yeast cells were harvested by centrifugation (500 X g/ 6 min), washed and suspended at the desired concentration in phosphate buffered saline (PBS).
by Bonferroni’s test were employed and significant differences were considered for P < 0.05. GraphPad PRISM (version 5) was used for graphic representations.
Culture of neutrophils
In order to determine possible alterations related to neutrophil activation, we evaluated cytokines and chemokines patterns produced by neutrophils from elderly and young DS comparing with age-matched controls. Our data showed that neutrophils from elderly and young DS individuals secreted higher IL-4 levels than their agematched control groups at 4 h and 18 h after co-culture with C. albicans (P
