Effect of Lactobacillus rhamnosus GR-1 and ... - Wiley Online Library

Microbiol Immunol 2009; 53: 487–495 doi:10.1111/j.1348-0421.2009.00154.x

ORIGINAL ARTICLE

Effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the ability of Candida albicans to infect cells and induce inflammation Rafael C.R. Martinez1 , Shannon L. Seney2 , Kelly L. Summers2,3 , Auro Nomizo1 , Elaine C.P. De Martinis1 and Gregor Reid2,3,4,5 1

Department of Clinical, Toxicological and Bromatological Analysis, Faculty of Pharmaceutical Sciences of Ribeirao ˜ Preto, Avenida do Cafe, ´ s/n, Campus of Ribeirao ˜ Preto, University of Sao ˜ Paulo, Ribeirao ˜ Preto, Sao ˜ Paulo 14040-903, Brazil 2 Lawson Health Research Institute and 5 Canadian Research & Development Centre for Probiotics, Room F2-116, St Joseph’s Health Care, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada 3 Department of Microbiology and Immunology and 4 Department of Surgery, University of Western Ontario, Richmond Street, London, Ontario, Canada

ABSTRACT Vulvovaginal candidiasis, a high prevailing infection worldwide, is mainly caused by Candida albicans. Probiotic Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 have been previously shown to be useful as adjuvants in the treatment of women with VVC. In order to demonstrate and better understand the anti-Candida activity of the probiotic microorganisms in an in vitro model simulating vaginal candidiasis, a human vaginal epithelial cell line (VK2/E6E7) was infected with C. albicans 3153a and then challenged with probiotic L. rhamnosus GR-1 and/or L. reuteri RC-14 or their respective CFS (alone or in combination). At each time point (0, 6, 12 and 24 hr), numbers of yeast, lactobacilli and viable VK2/E6E7 cells were determined and, at 0, 6 and 12 hr, the supernatants were measured for cytokine levels. We found that C. albicans induced a significant increase in IL-1α and IL-8 production by VK2/E6E7 cells. After lactobacilli challenge, epithelial cells did not alter IL-6, IL-1α, RANTES and VEGF levels. However, CFS from the probiotic microorganisms up-regulated IL-8 and IP-10 levels secreted by VK2/E6E7 cells infected with C. albicans. At 24 hr of co-incubation, L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 decreased the yeast population recoverable from the cells. In conclusion, L. reuteri RC-14 alone and together with L. rhamnosus GR-1 have the potential to inhibit the yeast growth and their CFS may up-regulate IL-8 and IP-10 secretion by VK2/E6E7 cells, which could possibly have played an important role in helping to clear VVC in vivo. Key words Candida, cytokine, Lactobacillus, probiotic, vulvovaginal candidiasis.

Vulvovaginal candidiasis is a condition that afflicts an estimated 75% of women at least once during their lifetime (1). It is predominantly caused by Candida albicans, a commensal dimorphic yeast-like fungal organism which

can be found in the normal vaginal environment and gastrointestinal tract (2). VVC results from overgrowth of Candida spp. and the microorganism is capable of destroying epithelial cells by direct invasion and release of

Correspondence Gregor Reid, Canadian Research & Development Centre for Probiotics, F2-116, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada. Tel: +519-646-6100 (ext. 65256); fax: +519-646-6031; email: [email protected] Received 24 November 2008; revised 23 March 2009; accepted 29 May 2009. List of Abbreviations: C. albicans, Candida albicans; CFS, cell-free supernatant; CFU, colony forming unit; IL, interleukin; IP-10, interferon-γ-inducible protein-10; KSFM, keratinocyte serum-free medium; L. reuteri RC-14, Lactobacillus reuteri RC-14; L. rhamnosus GR-1, Lactobacillus rhamnosus GR-1; MRS, de Man, Rogosa & Sharpe; NK, natural killer; PBS, phosphate-buffered saline; RANTES, regulated upon activation, normal T-cell expressed, and secreted; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; VLF, vaginal lavage fluid; VVC, vulvovaginal candidiasis.

c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

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substances such as prostaglandins and bradykinin which leads to vulvovaginal inflammation (vaginitis). This corresponds to the appearance of symptoms including vaginal soreness, irritation, vulvar burning, dyspareunia, and external dysuria (3–5). C. albicans can directly affect vaginal epithelial cells by skewing their cytokine production to a predominantly pro-inflammatory response (2). Furthermore, the release of chemoattractant cytokines (chemokines) by vaginal epithelial cells after contact with C. albicans is a critical mechanism for the induction of vaginitis, as chemokines recruit inflammatory cells into the environment thereby enhancing the release of inflammatory mediators such as soluble P-selectin, vascular adhesion molecule-1, CD14 and myeloperoxidase (6, 7). The pathogenesis of VVC has been thought to involve an impaired T-cell immune response against the invading Candida organisms (8). However, a recent human live challenge study (9) suggested that symptomatic VVC may instead be caused by an aggressive innate response to C. albicans arising from extensive neutrophil infiltration as opposed to a deficient T-cell response and, therefore, the presence of symptomatic infection appears to be more dependent on host factors than on properties of the organism. There is an urgent need for new treatments in the management of VVC, given the impact this condition has on the quality of life of hundreds of millions of women. Traditional anti-microbial azole derivates are effective in the treatment of primary acute VVC; however, they are less so at preventing recurrent attacks. In addition, in some patients, adverse effects such as gastrointestinal upset, headache, dizziness and rash may be caused by the use of fluconazole (10). Lactobacilli are predominant in the vagina of healthy premenopausal women and act to prevent the overgrowth of pathogens by producing acids, bacteriocins, biosurfactants, hydrogen peroxide and coaggregation molecules (11). Although lactobacilli and C. albicans often co-reside in the vagina of healthy women, clinical VVC can be triggered in some patients when antibiotic agents deplete lactobacilli flora and allow pathogen overgrowth (12, 13). Probiotics are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ (14) and may be a good option for use in preventing and/or co-treatment of VVC. They normally comprise organisms belonging to the genus Lactobacillus and Bifidobacterium (15). Jeavons (16) reviewed studies on the use of exogenous Lactobacillus for prevention and treatment of VVC. The author demonstrated that vaginally administered or orally ingested Lactobacillus is able to colonize the vaginal ecosystem and that controlled intervention studies regarding the effect of such colonization 488

on VVC are promising but few. Jeavons (16) also pointed out that the studies had a small number of participants, were inconsistent in the form of Lactobacillus used, and reported conflicting results. More recently, a double-blind, placebo-controlled clinical trial conducted by our group demonstrated the usefulness of probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 as a therapeutic adjuvant in the treatment of VVC. We showed that women who received a single dose of fluconazole (150 mg) and daily oral supplementation with probiotic L. rhamnosus GR-1 and L. reuteri RC-14 for 4 weeks exhibited significantly less vaginal discharge associated with symptoms of the condition (itching, burning, dyspareunia and dysuria) and there were also fewer vaginal samples positive for Candida sp. (based on the culture method) assessed on day 28 of treatment, when compared with patients receiving fluconazole and placebo capsules (17). The present study hypothesized that L. rhamnosus GR-1 and L. reuteri RC-14, known to modulate host immunity (18, 19), could interfere with the ability of C. albicans to grow on vaginal epithelial cell lines and induce inflammation.

MATERIALS AND METHODS Human epithelial cell line

VK2/E6E7 is an epithelial cell line derived from the vaginal mucosa of a healthy premenopausal female undergoing vaginal repair surgery that was subsequently immortalized with human papillomavirus 16/E6E7. Cells were maintained in KSFM (Invitrogen, Carlsbad CA, USA). Each ml of KSFM medium was supplemented with 50 μg bovine pituitary extract, 0.1 ng epidermal growth factor, and 100 U penicillin and streptomycin (Invitrogen) at 37◦ C with 5.0% CO 2 . Lactobacilli strains

Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 were grown anaerobically in MRS broth (Becton Dickinson, Sparks, MD, USA) at 37◦ C to reach 108 CFU/ml. To obtain CFS, cultures were centrifuged at 3000 g for 15 min at 4◦ C, and the supernatants were filtered through 0.20 μm filter units (Corning Incorporated, Corning, NY, USA) and neutralized to pH 7.0 (with 1 mol/l NaOH) before use. For experiments with lactobacilli cultures, cell pellets were resuspended in PBS, centrifuged once at 3000 g for 15 min at 4◦ C, and finally resuspended in KSFM with no antibiotics, before addition to the test wells (final concentration at 107 CFU/ml). c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

Anti-Candida effect of probiotic lactobacilli

Candida albicans strain

Candida albicans 3153a (ATCC 28367) was grown in brain heart infusion broth (Becton Dickinson) for 24 hr at 25◦ C to reach stationary phase. The blastoconidia were collected, washed with PBS, and enumerated using Trypan blue exclusion on a hemacytometer (Hausser Scientific; Horsham, PA, USA). In vitro model of human vaginal candidiasis and design of challenge study

The in vitro model of vaginal candidiasis was carried out as follows. Two days prior to challenge, 8.0 × 104 VK2/E6E7 cells/well were passed into a 24-well plate (Becton Dickinson) and grown to 90% confluency in 1 ml complete KSFM, checked by the use of an inverted microscope (Zeiss Axioskop, (Carl Zeiss, Göttingen, Germany). On the day of challenge (baseline, T = 0), supernatants from VK2/E6E7 cells were collected for cytokine analysis and baseline cell counts were obtained. For the remaining wells, the culture medium was aspirated, washed once with PBS, and replaced with 1 ml fresh KSFM lacking antibiotics per well. All conditions were carried out in triplicate wells. To the control wells, 10 ng/ml TNF-α was added. To all wells mimicking candidiasis infection, 10 μl C. albicans culture suspended in PBS was added to reach a final population of 2.0 × 104 CFU/ml (also representing 2.0 × 104 CFU/well) and incubated for 6 hr at 37◦ C (T = 6 hr). After 6 hr, the cells were challenged with: (i) L. rhamnosus GR-1 (107 CFU/ml); (ii) CFS (1:10 v/v) from L. rhamnosus GR-1; (iii) L. reuteri RC-14 (107 CFU/ml); (iv) CFS (1:10 v/v) from L. reuteri RC14; (v) both L. rhamnosus GR-1 and L. reuteri RC-14 (107 CFU/ml each); and (vi) CFS from both L. rhamnosus GR-1 and L. reuteri RC-14 (1:10 v/v). After challenge, wells were incubated for an additional 6 hr (T = 12 hr) and then supernatants were harvested and used for assessing VK2/E6E7, lactobacilli and yeast populations, and then frozen for cytokine analysis. Duplicate plates were incubated for a further 12 hr time point (T = 24 hr) and supernatants harvested as mentioned above. Determination of VK2/E6E7 cells, C. albicans and lactobacilli populations

Populations of VK2/E6E7 cells were determined from one well of every condition at T = 0, 6 hr, 12 hr, and 24 hr using Trypan blue exclusion on a hemacytometer (Hausser Scientific). Total C. albicans 3153a and/or L. rhamnosus GR-1 and L. reuteri RC-14 counts were assessed at T = 0, 6 hr, 12 hr, and 24 hr. For the two other wells regarding every condition tested in triplicate, Triton X-100 (Sigma-Aldrich Canada, Oakville, Ontario, Canada) was added to a final c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

concentration of 0.5% (v/v) and incubated for 15 min until VK2/E6E7 cells were completely lysed. Supernatants were collected, serially diluted in sterile PBS and plated on either R Candida (Oxoid, Basingstoke, Hampshire, Chromagar UK) and incubated for 48 hr at 37◦ C under aerobic conditions or on MRS containing 200 μg/ml miconazole and incubated for 48 hr at 37◦ C under anaerobic conditions for determination of yeast and lactobacilli populations (respectively), or on both media depending on the condition tested. After plating, the supernatants were centrifuged at 12 000 g for 5 min, transferred to a new tube and stored at −80◦ C until analysis. Supernatant collection for examination of cytokines and chemokines

Frozen supernatants taken at T = 0, 6 hr, and 12 hr were tested for the following cytokines: IL-6, IL-8, IL-1α, IP-10, RANTES and VEGF. Levels of cytokines were measured using multiplexed biomarker immunoassay kits according to the manufacturer’s instructions (LINCOplex; Millipore Corp., Billerica, MA, USA). A Bio-PlexTM 200 readout system was used (Bio-Rad Laboratories, Hercules, CA, USA), R xMAPTM fluorescent bead-based which uses Luminex technology (Luminex Corp., Austin, TX, USA). Cytokine levels (pg/ml) were automatically calculated from standard curves using Bio-Plex Manager software (v. 4.1.1; Bio-Rad). Statistical analysis

Data presented are the mean of two independent replicates ± standard deviation. Unpaired Student’s t-test was used to evaluate differences among cytokines constitutively produced by VK2/E6E7 cells and that induced by co-cultivation with C. albicans 3153a. Significant differences were defined as having a P value < 0.05. In order to study the effect of Lactobacillus and their CFS (alone or in combination) against VK2/E6E7 cells (or VK2/E6E7 infected with C. albicans 3153a), one-way ANOVA with a level of significance set at P < 0.05 was used. Whenever statistically significant differences were found, the Bonferroni test was carried out and the critical level was set at P < 0.05. Sigma Stat 2.03 (Systat Software Inc., San Jose, CA, USA) was used for all the tests.

RESULTS In the present study, at baseline (T = 0), levels of cytokine production by VK2/E6E7 cells were 57.17 pg/ml (IL-8), 28.44 pg/ml (IL-1α), 113.36 pg/ml (VEGF), 4.45 pg/ml (IL-6), 12.43 pg/ml (IP-10) and 16.20 pg/ml (RANTES). After 6 hr of incubation, increased levels of IL-1α (107.39 pg/ml) and VEGF (52.71 pg/ml), but lower 489


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Cytokine Fig. 1. Production of IL-6, IL-8, IL-1α, IP-10, RANTES and VEGF (expressed as pg/ml) by epithelial cell line VK2/E6E7 cultivated alone, grown with 10 ng/ml TNF-α, and infected with C. albicans 3153a. Cytokine levels were measured after (a) 6 hr and (b) 12 hr of incubation. TNF-α

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was used as a positive control. ∗ Cytokine levels are significantly different (P < 0.05) than VK2/E6E7 cultivated alone. Combined mean values from two independent experiments are shown.



VK2/E6E7 + C. albicans VK2/E6E7 + C. albicans + L. rhamnosus GR1 VK2/E6E7 + C. albicans + L. rhamnosus GR1 (CFS) VK2/E6E7 + C. albicans + L. reuteri RC-14 VK2/E6E7 + C. albicans + L. reuteri RC-14 (CFS) VK2/E6E7 + C. albicans + L. rhamnosus GR-1 + L. reuteri RC-14 VK2/E6E7 + C. albicans + L. rhamnosus GR-1 (CFS) + L. reuteri RC-14 (CFS)

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Cytokine Fig. 2. Production of IL-8, IL-1α and IP-10 (expressed as pg/ml) by epithelial cell line VK2/E6E7 infected with C. albicans 3153a, not challenged or challenged with: (i) L. rhamnosus GR-1 (107 CFU/ml); (ii) CFS from L. rhamnosus GR-1 (1:10 v/v); (iii) L. reuteri RC-14 (107 CFU/ml); (iv) CFS from L. reuteri RC-14 (1:10 v/v); (v) both L. rhamnosus GR-1 and L. reuteri

RC-14 (107 CFU/ml each); and (vi) CFS from both L. rhamnosus GR-1 and L. reuteri RC-14 (1:10 v/v). Cytokine levels were measured after 12 hr of incubation. ∗ Cytokine levels are significantly different (P < 0.05) than VK2/E6E7 cocultivated with C. albicans 3153a. Combined mean values from two independent experiments are shown.

levels of IL-8 (15.33 pg/ml), RANTES (6.60 pg/ml), IP10 (4.64 pg/ml), and IL-6 (4.54 pg/ml) were observed (Fig. 1a). Comparatively, after 6 hr of co-incubation with TNF-α, a known activator of VK2/E6E7 cells (2), an upregulation in the production of IL-6 (2.0-fold), IL-8 (5.5fold) and IP-10 (2.0-fold) by VK2/E6E7 cells was observed (P < 0.05). However, no increase in the levels of IL-1α, RANTES and VEGF was observed by TNF-α stimulation (P > 0.05; Fig. 1a). After 12 hr, TNF-α stimulation up-regulated the production of all the six cytokines by VK2/E6E7 cells; however, only for IL-8 and IP-10 (4-fold increase and 3.5-fold increase, respectively) was there a statistical difference (P < 0.05) when compared to the vaginal cells cultivated alone (Fig. 1b). VK2/E6E7 numbers remained stable at 8.0 × 104 cells/well up to 6 hr culture in the presence of C. albicans 3153a. After 12 hr culture, C. albicans affected VK2/E6E7 cell viability as numbers decreased to 1.0 × 104 cells/well. The yeast stimulated a dramatic increase in IL-1α secretion by VK2/E6E7 cells at T = 6 hr (3-fold; P < 0.05;

Fig. 1a) and T = 12 hr (31-fold; P < 0.05; Fig. 1b). IL-8 secretion by VK2/E6E7 cells was increased by C. albicans infection only after 12 hr coculture (Fig. 1b). C. albicans had no effect on IL-6, IP-10, RANTES and VEGF levels secreted by VK2/E6E7 as verified at T = 6 hr (Fig. 1a) and T = 12 hr (Fig. 1b). When VK2/E6E7 vaginal epithelial cells grown with C. albicans were challenged with cells or CFS from L. reuteri RC-14 or L. rhamnosus GR-1 (alone or in combination), results assessed at T = 12 hr showed that only CFS from L. reuteri RC-14 alone or in combination with CFS from L. rhamnosus GR-1 induced an increase in the production of IL-8 by the vaginal cells (P < 0.05; Fig. 2). Furthermore, VK2/E6E7 cells infected with C. albicans secreted higher amounts of IP-10 when challenged with CFS from L. rhamnosus GR-1 alone and in combination with CFS from L. reuteri RC-14 (P < 0.05; Fig. 2). None of the tested conditions affected production of IL-6, IL-1α, RANTES and VEGF (P > 0.05; data not shown).


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Cytokine Fig. 3. Production of IL-8 and IP-10 (expressed as pg/ml) by epithelial cell line VK2/E6E7 in the presence of: (i) L. rhamnosus GR-1 (107 CFU/ml); (ii) CFS from L. rhamnosus GR-1 (1:10 v/v); (iii) L. reuteri RC-14 (107 CFU/ml); (iv) CFS from L. reuteri RC-14 (1:10 v/v); (v) both L. rhamnosus GR-1 and L. reuteri RC-14 (107 CFU/ml each); and (vi) CFS from both L. rhamnosus

GR-1 and L. reuteri RC-14 (1:10 v/v). Cytokine levels were measured after 12 hr of incubation. ∗ Cytokine levels are significantly different (P < 0.05) than VK2/E6E7 cultivated alone. Combined mean values from two independent experiments are shown.

Cells or CFS from L. rhamnosus GR-1 and L. reuteri RC-14 (alone or in combination) were added to wells with VK2/E6E7 cells and measurements of IL-8 assessed at T = 12 hr indicated that the production of the cytokine was up-regulated under all those conditions (P > 0.05; Fig. 3). Furthermore, L. reuteri RC-14 was also able to increase the IP-10 level secreted by the vaginal epithelial cell line (P < 0.05; Fig. 3). In our experiments, lactobacilli populations assessed at T = 0, 6 hr, 12 hr, and 24 hr remained stable at 107 CFU/ml. C. albicans populations assessed at T = 6 hr and 12 hr were similar in all conditions tested (P < 0.05; Fig. 4). However, at T = 24 hr, population of C. albicans cultured alone (control) reached 4.0 × 106 CFU/ml (6.6 log CFU/ml) compared to only 3.0 × 105 CFU/ml (5.5 log CFU/ml) assessed in the presence of L. reuteri RC-14 alone or in combination with L. rhamnosus GR-1 (P < 0.05; Fig. 4).

ing to our study, VK2/E6E7 cells constitutively secrete a range of six cytokines, including IL-6, IL-8, IL-1α, IP-10, RANTES and VEGF. This supports other studies which showed that VK2/E6E7 can spontaneously secrete IL-8 (2, 25), IL-1α and IL-6 (2). We have demonstrated that TNF-α was able to upregulate the production of IL-8 and IP-10 assessed at both T = 6 hr and T = 12 hr, whereas IL-6 was only up-regulated at T = 6 hr and RANTES at T = 12 hr (Fig. 1a,b). IL-1α and VEGF (a cytokine known for regulating angiogenesis, increasing microvascular permeability and recruiting monocytes and macrophages (26)) were not up-regulated by the addition of TNF-α at T = 6 hr or T = 12 hr. Our findings support a previous study that showed increased secretion of IL-6 (a chemoattractant cytokine, important for the recruitment of inflammatory cells and able to modulate the function of many types of immune cells (27)) by TNF-α-stimulated VK2/E6E7 cells (2). Fichorova and Anderson (25) reported that VK2/E6E7 cells stimulated with 0.1 μg/ml TNF-α also showed variable increases in IL-6, IL-8 and RANTES (a potent chemoattractant factor for monocytes and T cells (28)).

DISCUSSION Epithelial cells play a key role in mediating mucosal immune responses including antigen presentation, antimicrobial activity and cytokine production (20–24). Accord492



VK2/E6E7 + C. albicans VK2/E6E7 + C. albicans + L. rhamnosus GR1 VK2/E6E7 + C. albicans + L. rhamnosus GR1 (CFS) VK2/E6E7 + C. albicans + L. reuteri RC14 VK2/E6E7 + C. albicans + L. reuteri RC14 (CFS) VK2/E6E7 + C. albicans + L. rhamnosus GR1 + L. reuteri RC14 VK2/E6E7 + C. albicans + L. rhamnosus GR1 (CFS) + L. reuteri RC14 (CFS)

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Time (h) Fig. 4. Enumeration (log CFU/ml) of C. albicans 3153a at 6 hr, 12 hr, and 24 hr of incubation at 37◦ C on 24-well plates containing epithelial cell line VK2/E6E7, alone or challenged with: (i) L. rhamnosus GR-1 (107 CFU/ml); (ii) CFS (1:10 v/v) from L. rhamnosus GR-1; (iii) L. reuteri RC-14 (107 CFU/ml); (iv) CFS (1:10 v/v) from L. reuteri RC-14; (v) both L. rhamnosus GR-1 and L. reuteri RC-14 (107 CFU/ml each); and (vi) CFS

(1:10 v/v) from both L. rhamnosus GR-1 and L. reuteri RC-14. Populations of C. albicans 3153a assessed at T = 24 hr for conditions (iii) and (iv) are statistically different (P < 0.05) when compared to those obtained with the yeast strain cultured alone. Combined mean values from two independent experiments are shown.

The present study demonstrates that C. albicans stimulates production by VK2/E6E7 cells of the proinflammatory cytokines IL-8 and IL-1α (Fig. 2). This supports previous reports demonstrating that IL-1 plays an active role in the immune response to C. albicans infection (2, 29). Although Steele and Fidel (2) have used the same in vitro model of vaginal candidiasis and also demonstrated an increase in IL-1α secretion by the epithelial cell line VK2/E6E7 infected with C. albicans, the time points they analyzed were later (24 hr and 96 hr) when compared to ours (6 hr and 12 hr). Steele and Fidel (2) demonstrated little to no Candida-specific production of IL-8 by VK2/E6E7 cells, whereas the present study showed an IL-8 response to the yeast within 12 hr. IL-8 and the IL-1 family of cytokines are well-known mediators of both local and systemic inflammation (30). Fan and coworkers (31) evaluated the VLF from three groups of women and observed that the concentration of IL-8 was increased in VVC samples, and it was highest for subjects with severe VVC. IL-8 is considered important for recruiting neutrophils to areas of inflammation and/or

infection in vivo (6, 32–34) and its measurement is useful as a biomarker of antimicrobial response at mucosal sites, including the female genital tract (35). In the present study, CFS from L. reuteri RC-14 in combination with CFS from L. rhamnosus GR-1 increased both IL-8 and IP-10 production by VK2/E6E7 infected with C. albicans, whereas CFS from L. reuteri RC-14 (alone) up-regulated the level of IL-8, and CFS from L. rhamnosus GR-1 stimulated the secretion of IP-10 (Fig. 2). In all these cases, the effects may be due to any soluble compound actively produced by the lactobacilli strains and the mechanisms evolved remain to be determined. Furthermore, cells or CFS from L. rhamnosus GR-1 and L. reuteri RC-14 (alone or in combination) were shown to up-regulate the release of IL-8 when in direct contact with VK2/E6E7 cells, whereas only L. reuteri RC-14 stimulated the production of IP-10 (Fig. 4). IP-10 is a chemokine with chemoattractant properties towards Th1 lymphocytes and NK cells and it most likely plays a key role in host defense against Candida infection (36). Therefore, the effects observed on the secretion of cytokines by VK2/E6E7 cells infected


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with C. albicans after challenge with CFS from L. rhamnosus GR-1 and/or L. reuteri RC-14 may function to attract more immunological cells to the locus of the infection and help to solve it in a faster way. L. reuteri RC-14 (alone) and in combination with L. rhamnosus GR-1 was able to inhibit the growth of C. albicans as determined at 24 hr of incubation at 37◦ C (Fig. 4). Using this scenario, we hypothesized that competition for nutrients between both microorganisms may have played an important role and that the presence of C. albicans cells may be required to trigger (or even increase) the production of anti-Candida compounds by the lactobacilli strains. L. rhamnosus GR-1 and L. reuteri RC-14 were previously demonstrated to inhibit the growth of C. albicans by the over-layer agar method (37). Furthermore, L. rhamnosus GR-1 is able to inhibit the yeast biofilm formation, and this anti-Candida effect is possibly due to down-regulation of genes involved in filamentation, an important factor for candidal virulence and biofilm formation (38). In summary, among six cytokines tested here (IL-6, IL8, IL-1α, IP-10, RANTES and VEGF), only IL-8 and IP-10 were up-regulated by challenge with CSF from the probiotic microorganisms L. rhamnosus GR-1 and L. reuteri RC-14. Also, L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 was effective in suppressing C. albicans growth and spread on vaginal cells. Together, these may be important to help clear the infection in vivo (as demonstrated in the previous clinical study conducted by our group with women diagnosed with VVC (17)). However, more studies are required to better understand the exact mechanisms involved and help explain the positive outcome.

ACKNOWLEDGMENTS R. C. R. Martinez is grateful to São Paulo State Foundation for Support of Science (FAPESP, Process #04/14580-0) and to the Brazilian Post Graduate Federal Agency (CAPES, Process #6159/06-2) for supporting his PhD studies in Brazil and Canada, respectively. The authors acknowledge Dr Peter Cadieux from the University of Western Ontario (London, Ontario, Canada), who kindly donated the VK2/E6E7 epithelium cell line. Assistance from a grant from Natural Sciences and Engineering Research Council of Canada (NSERC) is appreciated. REFERENCES 1. Sobel J.D., Faro S., Force R.W., Foxman B., Ledger W.J., Nyirjesy P.R., Reed B.D., Summers P.R. (1998) Vulvovaginal candidiasis: epidemiologic, diagnostic, and therapeutic considerations. Am J Obstet Gynecol 178: 203–11. 2. Steele C., Fidel P.L. Jr. (2002) Cytokine and chemokine production

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